Constitutive Activation of Integrin α9 Augments Self-Directed Hyperplastic and Proinflammatory Properties of Fibroblast-like Synoviocytes of Rheumatoid Arthritis.

Despite advances in the treatment of rheumatoid arthritis (RA), currently approved medications can have significant side effects due to their direct immunosuppressive activities. Additionally, current therapies do not address residual synovial inflammation. In this study, we evaluated the role of integrin α9 and its ligand, tenascin-C (Tn-C), on the proliferative and inflammatory response of fibroblast-like synoviocytes (FLSs) from RA patients grown in three-dimensional (3D)-micromass culture. FLSs from osteoarthritis patients, when grown in the 3D-culture system, formed self-directed lining-like structures, whereas FLSs from RA tissues (RA-FLSs) developed an abnormal structure of condensed cellular accumulation reflective of the pathogenic features of RA synovial tissues. Additionally, RA-FLSs grown in 3D culture showed autonomous production of proinflammatory mediators. Predominant expression of α9 and Tn-C was observed in the condensed lining, and knockdown of these molecules abrogated the abnormal lining-like structure formation and suppressed the spontaneous expression of matrix metalloproteinases, IL-6, TNFSF11/RANKL, and cadherin-11. Disruption of α9 also inhibited expression of Tn-C, suggesting existence of a positive feedback loop in which the engagement of α9 with Tn-C self-amplifies its own signaling and promotes progression of synovial hyperplasia. Depletion of α9 also suppressed the platelet-derived growth factor-induced hyperplastic response of RA-FLSs and blunted the TNF-α-induced expression of matrix metalloproteinases and IL-6. Finally, α9-blocking Ab also suppressed the formation of the condensed cellular lining by RA-FLSs in 3D cultures in a concentration-related manner. This study demonstrates the central role of α9 in pathogenic behaviors of RA-FLSs and highlights the potential of α9-blocking agents as a nonimmunosuppressive treatment for RA-associated synovitis.

Preclinical Characterization of ASP2713, a Novel Igß and FcγRIIB Cross-Linking Antibody, for Prediction of Human Pharmacokinetics and Clinically Effective Dose.

Previously, we reported the fundamental pharmacological characteristics of a novel Igß and Fc gamma receptor IIB cross-linking antibody, ASP2713, as a new treatment option for systemic lupus erythematosus. The aims of the present study were to investigate ASP2713's characteristics with regard to pharmacological effect, pharmacokinetics (PK), and receptor occupancy, and to predict its human PK and clinically effective dose. The relationship between the concentration and receptor occupancy of ASP2713 for Igß of B cell receptors was examined using whole blood B cells. Calculated EC50 values in cynomolgus monkeys and healthy volunteers were 0.35 and 0.058 µg/mL, respectively. Dose-dependent inhibition of anti-tetanus toxoid (TTx) antibody production, PK, and receptor occupancy of ASP2713 in TTx-sensitized cynomolgus monkeys suggested a minimally effective dose of 1 mg/kg by single intravenous (IV) administration. Scaling-up of monkey PK parameters to humans by allometric scaling predicted a clinically effective dose of 0.4 mg/kg IV administration at 4-week intervals to maintain a trough concentration in humans which achieved the same receptor occupancy expected at the effective dose in monkeys. This study aids in understanding the characteristics of ASP2713 and can be used as a basis for clinical dose setting.

A novel monoclonal antibody cross-reactive with both human and mouse α9 integrin useful for therapy against rheumatoid arthritis.

This study introduces a novel monoclonal anti-α9 integrin antibody (MA9-413) with human variable regions, isolated by phage display technology. MA9-413 specifically binds to both human and mouse α9 integrin by recognizing a conserved loop region designated as L1 (amino acids 104-122 of human α9 integrin). MA9-413 inhibits human and mouse α9 integrin-dependent cell adhesion to ligands and suppresses synovial inflammation and osteoclast activation in a mouse model of arthritis. This is the first monoclonal anti-α9 integrin antibody that can react with and functionally inhibit both human and mouse α9 integrin. MA9-413 allows data acquisition both in animal and human pharmacological studies without resorting to surrogate antibodies. Since MA9-413 showed certain therapeutic effects in the mouse arthritis model, it can be considered as a useful therapy against rheumatoid arthritis and other α9 integrin-associated diseases.

Osteopontin is dispensable for protection against high load systemic fungal infection.

Osteopontin (OPN) is a multi-functional cytokine which is involved in the pathogenesis of autoimmune disease. We previously reported that thrombin-cleaved form of OPN plays a pathogenic role in murine model of rheumatoid arthritis (RA) by using neutralizing antibody (M5) reacting against the cryptic epitope within OPN, exposed by thrombin cleavage of OPN. It has been shown that OPN-deficient mice are susceptible to various infections, demonstrating the protective role of OPN against various infectious diseases. However, it remains to be clarified whether and how OPN is involved in protection against systemic fungal infection. In a murine model of systemic fungal infection, OPN-deficient mice showed the increase in the susceptibility to low load, but not to high load fungal infection, indicating the protective of OPN against mild or severe forms of infections. However, mice treatment with M5 antibody did not alter the susceptibility to both high and low load fungal infection. These experiments suggest that in sharp contrast to the complete abrogation of OPN expression in OPN-deficient mice, the neutralization of OPN by antibody against thrombin-cleaved form of OPN does not interfere with the host defense against high and low load fungal infection. These findings suggest that the neutralizing antibody which is specific for the epitope of thrombin-cleaved OPN may become an attractive therapeutic means for the treatment of RA without interfering host defense system.

Integrin, alpha9 subunit blockade suppresses collagen-induced arthritis with minimal systemic immunomodulation.

Integrin, alpha9 subunit (hereinafter, alpha9) has been identified as a novel putative therapeutic target for rheumatoid arthritis (RA). Support for this target comes from the observations that alpha9 is overexpressed both in the joints of RA patients and in animal models of arthritis. In the experimental models, the increase in alpha9 expression precedes the onset of arthritic symptoms. The current study presents data on the pharmacological profile of an anti-alpha9 antibody in a collagen-induced arthritis (CIA) mouse model. Administration of an alpha9-blocking antibody in CIA mice suppressed the development of arthritis and significantly decreased plasma level of activated fibroblast-like synoviocyte (FLS)-derived biomarkers without reducing the formation of anti-type II collagen antibodies. While anti-alpha9 antibody administration significantly suppress the accumulation of immune cells in arthritic joints it had no effect on immune cell number in the spleen. Furthermore, in non-arthritic mice, alpha9 had no inhibitory effect in either a mixed lymphocyte reaction (MLR) or in a delayed type hypersensitivity (DTH) reaction. These results suggest that blocking alpha9 exerts its anti-arthritic effect through suppression of FLS-activation via a non-immune mediated mechanism. Finally, therapeutic administration of anti-alpha9 antibody alleviated established arthritis in CIA mice. Our data provide evidence that alpha9 blockade is a promising therapy for joint inflammation with minimal systemic immunomodulation.

Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis.

It has been shown that osteopontin (OPN) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). However, the molecular mechanism of OPN action is yet to be elucidated. Splenic monocytes obtained from arthritic mice exhibited a significant capacity for cell migration toward thrombin-cleaved OPN but not toward full-length OPN. Migratory monocytes expressed alpha9 and alpha4 integrins. Since cleavage of OPN by thrombin exposes the cryptic epitope recognized by alpha9 and alpha4 integrins, we investigated the role of the cryptic epitope SLAYGLR in a murine RA model by using a specific antibody (M5) reacting to SLAYGLR sequence. The M5 antibody could abrogate monocyte migration toward the thrombin-cleaved form of OPN. Importantly, M5 antibody could inhibit the proliferation of synovium, bone erosion, and inflammatory cell infiltration in arthritic joints. Thus, we demonstrated that a cryptic epitope, the SLAYGLR sequence of murine OPN, is critically involved in the pathogenesis of a murine model of RA.

Successful treatment of collagen-induced arthritis in non-human primates by chimeric anti-osteopontin antibody.

The presence of thrombin-cleaved form of osteopontin well correlated with various inflammatory disease activities in not only rodents, but also humans. We previously demonstrated that the blocking of the interaction of a cryptic epitope within osteopontin, which is exposed by thrombin cleavage, with its integrins by specific antibody recognizing cryptic epitope of mouse osteopontin, could significantly inhibits the development of arthritis in mice. We generated a murine monoclonal antibody, 2K1, specifically recognizing a cryptic epitope of human osteopontin, SVVYGLR. We constructed a chimeric antibody, C2K1 in which variable region of 2K1 was fused with human IgG1 constant region. In the present study, we investigated whether the therapeutic administration of C2K1 could ameliorate the established collagen-induced arthritis in cynomolgus monkey. Thus, C2K1 was injected after the onset of arthritis. The inhibition of joint swelling by C2K1 became evident at 4 to 5 weeks after initiation of arthritis, when blood level of C2K1 was peaked. Joint swelling reappeared along with the sharp decline of C2K1 blood levels at 6 weeks. Importantly, destruction of bone and cartilage in joints was still significantly prevented at 10 weeks when blood level of C2K1 was quite low if any and anti-C2K1 antibody emerged. These results demonstrate that neutralizing antibody against the cryptic epitope of osteopontin can be a future therapeutic choice for patients with rheumatoid arthritis.

Decreased AP-1 activity and interleukin-11 expression by bone marrow stromal cells may be associated with impaired bone formation in aged mice.

UNLABELLED: Expression of an osteogenic cytokine, IL-11, is decreased in SAMP6. We show here that IL-11 transcription largely depends on AP-1 transcription factors, activities of which are decreased in SAMP6 as well as aged ICR mice. Therefore, diminished AP-1 activity and the resultant decline in IL-11 expression may play a role in impaired bone formation in the aged. INTRODUCTION: Evidence suggests that impaired osteoblastogenesis contributes to aging-associated osteopenia. The P6 strain of senescence-accelerated mice (SAM) is an animal model of senile osteoporosis, which exhibits low bone mass caused by impaired bone formation. Bone marrow stromal cells from SAMP6 show decreased osteoblastogenesis and increased adipogenesis. We previously demonstrated that these abnormalities of SAMP6 stromal cells may be attributed to decreased expression of interleukin (IL)-11. METHODS: In this study, we attempted to determine the molecular mechanism of decreased IL-11 expression by SAMP6 stromal cells by cloning and analyzing the mouse IL-11 gene promoter. RESULTS AND CONCLUSIONS: We found that two tandem activating protein-1 (AP-1) sites that reside immediately upstream of TATA box play critical roles in IL-11 gene transcription. Gel shift analysis showed that binding activity to the IL-11 AP-1 sites was reduced in SAMP6 stromal cell nuclear extracts. Among multiple components of AP-1 transcription factors, Jun D binding was particularly decreased. Furthermore, decreased Jun D binding and IL-11 expression by stromal cells was also observed in aged mice of the ICR strain. Therefore, decreased AP-1 activity and a resultant decline in IL-11 expression by bone marrow stromal cells may play a role in impaired bone formation in the aged.

Prevention of the cryptic epitope SLAYGLR within osteopontin does not influence susceptibility to Candida albicans infection.

The effect of an antiosteopontin antibody (M5 Ab) reacting with the SLAYGLR sequence within osteopontin on the host susceptibility to infection was investigated in a murine model of disseminated candidiasis. The treatment with anti-tumor necrosis factor alpha antibody enhanced fungal infection, while the treatment with M5 Ab did not affect the infection.