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Objectives To evaluate the long-term safety and efficacy of E6011, a humanized anti-fractalkine monoclonal antibody, in patients with rheumatoid arthritis (RA) with an inadequate response to methotrexate (MTX). Methods Active RA patients with an inadequate response to MTX were randomly assigned to the E6011 or placebo group and received the study drug subcutaneously every 2 weeks during a 24-week double-blind study period. Subjects who completed evaluations at Week 24 were rolled over into the extension phase and received open-label E6011 (200 or 400 mg) every 2 weeks until Week 102. The safety analysis was conducted up to Week 104, and the efficacy analysis was conducted up to Week 84. Results A total of 169 subjects completed the double-blind treatment phase and were rolled over into the extension phase. In total, 167 (98.8%) subjects experienced any adverse events, and the incidence of treatment-related adverse events was 56.2%. The American College of Rheumatology 20 response rates were observed between 40 and 70% during the extension phase. Conclusions E6011 was safe and well tolerated with no notable safety concerns up to 102 weeks in RA patients with an inadequate response to MTX.

Objectives The objective of the study is to evaluate the long-term safety and efficacy of E6011, a humanized anti-fractalkine monoclonal antibody, in patients with rheumatoid arthritis with an inadequate response to biological disease-modifying antirheumatic drugs. Methods In the double-blind treatment phase (24 weeks), placebo or E6011 400 mg was administered until Week 10. Thereafter, E6011 200 mg or 400 mg was administered to Week 22. Subjects who completed the evaluation at Week 24 of the treatment phase were rolled over into the extension phase. The extension phase lasted until Week 104, and all subjects received E6011 400 mg or 200 mg every 2 weeks in an open-label manner until Week 102. Results A total of 47 subjects completed the double-blind treatment phase and were rolled over into the extension phase. In total, 46 (97.9%) subjects experienced any adverse events, and the incidence of treatment-related adverse events was 57.4%. No clear efficacy trend in the American College of Rheumatology 20% response rates was observed. Conclusions E6011 was well tolerated in active rheumatoid arthritis patients who had shown an inadequate response to biologic disease-modifying antirheumatic drugs, but no clear benefit in the American College of Rheumatology 20% response rates was observed. Further studies are needed to clarify the clinical benefit of E6011.

Background and Aim E6011 is a humanized monoclonal antibody targeting fractalkine (FKN), a CX3C chemokine, which regulates leukocyte trafficking during inflammation. We evaluated the safety and pharmacokinetic profile of E6011 in patients with Crohn's disease (CD) and also performed preliminary pharmacodynamic (PD) and efficacy assessments. Methods This study included a 12‐week multiple ascending dose (MAD) phase (2, 5, 10, and 15 mg/kg intravenously every 2 weeks, n = 6, 8, 7, and 7, respectively) and a 40‐week Extension phase (n = 12) at the same dose as the MAD phase. Serum E6011, serum total FKN (free soluble FKN and E6011‐FKN complex) as a PD marker and CD activity index were evaluated. The primary outcome was safety assessment in the MAD phase. Results Twenty‐seven (96%) of 28 patients had previously been treated with anti‐tumor necrosis factor α agents. During the MAD phase, adverse events (AEs) occurred in 18 (64%). The most common AE was nasopharyngitis (five patients, 18%). No severe AEs occurred. Serious AEs occurred in three patients, progression of CD in two, and anemia in one. Serum E6011 concentrations increased dose‐dependently after infusion and reached a plateau around 4–6 weeks. Serum total FKN rose simultaneously. Five (18%) patients developed anti‐E6011 antibodies during the study. Overall, clinical response and clinical remission were observed at Week 12 in 40% (10/25) and 16% (4/25) of active CD patients, respectively. Conclusion E6011 was well‐tolerated and might be effective in CD patients. These findings need to be clarified in a randomized controlled study.

To evaluate the safety and efficacy of E6011, a novel humanized anti-fractalkine monoclonal antibody, in patients with active rheumatoid arthritis (RA) with an inadequate response to biological disease-modifying antirheumatic drugs (DMARDs).Active RA patients inadequately responding to biological DMARDs were randomly assigned to placebo or E6011 400-mg group at a 1:1 ratio, and administered E6011 at weeks 0, 1, 2, and subsequently every 2 weeks. Primary endpoint was American College of Rheumatology (ACR)20 response at week 12.Of 64, 33 received placebo, 31 received E6011 400-mg. The ACR20 response rate at week 12 (non-responder imputation) was 27.3% and 22.6% in the placebo and E6011 groups, respectively. ACR50, ACR70 response rates at week 12 were 3.0%, 0% in the placebo and 9.7%, 3.2% in the E6011 group. Exploratory PK exposure analysis revealed that the effect of E6011 tended to be clearer in patients with higher serum trough E6011 concentration. E6011 was well tolerated with no notable safety concerns.E6011 400-mg was well tolerated but had no clear efficacy at week 12 in RA patients with inadequate response to biologics. Further investigations are warranted to determine the optimal clinical dose and evaluation period for E6011.

OBJECTIVE To assess the preclinical efficacy and mechanism of action of an anti-CX3 CL1 monoclonal antibody (mAb) in systemic sclerosis (SSc). METHODS Cultured human dermal fibroblasts were used to evaluate the direct effect of anti-CX3 CL1 mAb on fibroblasts. In addition, bleomycin-induced and growth factor-induced models of SSc were used to investigate the effect of anti-CX3 CL1 mAb on leukocyte infiltration, collagen deposition, and vascular damage in the skin. RESULTS Anti-CX3 CL1 mAb treatment significantly inhibited Smad3 phosphorylation (P < 0.05) and expression of type I collagen and fibronectin 1 (P < 0.01) in dermal fibroblasts stimulated with transforming growth factor β1 (TGFβ1). In the bleomycin model, daily subcutaneous bleomycin injection increased serum CX3 CL1 levels (P < 0.05) and augmented lesional CX3 CL1 expression. Simultaneous administration of anti-CX3 CL1 mAb or CX3 CR1 deficiency significantly suppressed the dermal thickness, collagen content, and capillary loss caused by bleomycin (P < 0.05). Injection of bleomycin induced expression of pSmad3 and TGFβ1 in the skin, which was inhibited by anti-CX3 CL1 mAb. Further, the dermal infiltration of CX3 CR1+ cells, macrophages (inflammatory and alternatively activated [M2-like] subsets), and CD3+ cells significantly decreased following anti-CX3 CL1 mAb therapy (P < 0.05), as did the enhanced skin expression of fibrogenic molecules, such as thymic stromal lymphopoietin and secreted phosphoprotein 1 (P < 0.05). However, the treatment did not significantly reduce established skin fibrosis. In the second model, simultaneous anti-mCX3 CL1 mAb therapy significantly diminished the skin fibrosis induced by serial subcutaneous injection of TGFβ and connective tissue growth factor (P < 0.01). CONCLUSION Anti-CX3 CL1 mAb therapy may be a novel approach for treating early skin fibrosis in inflammation-driven fibrotic skin disorders such as SSc.

Objective To evaluate the efficacy and safety of E6011, a humanized IgG2 monoclonal antibody against human fractalkine (FKN), in a phase II, double-blind, placebo-controlled study in rheumatoid arthritis (RA) patients. Methods Patients with moderate-to-severe RA who had an inadequate response to methotrexate were randomly assigned to a placebo group or to E6011 100-mg, 200-mg, or 400/200-mg groups at a 2:1:2:2 ratio. During the 24-week period, patients received the study drug subcutaneously at weeks 0, 1, and 2 and then once every 2 weeks. The primary end point was the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12. Results Study drugs were administered to 190 patients (placebo, n = 54; E6011 100 mg, n = 28; E6011 200 mg, n = 54; E6011 400/200 mg, n = 54), and 169 patients completed treatment. A significant difference from placebo was not found in ACR20 response rates at week 12 (37.0% [placebo], 39.3% [100 mg], 48.1% [200 mg], and 46.3% [400/200 mg], using nonresponder imputation). As a secondary end point, ACR20 response rate in the 200-mg and 400/200-mg groups attained statistical significance at week 24 (35.2% [placebo], 39.3% [100 mg], 53.7% [200 mg], and 57.4% [400/200 mg]). Subsequent exploratory subgroup analysis revealed greater efficacy of E6011, particularly in patients with a higher baseline proportion of CD16+ monocytes; ACR20 response rates in this patient subgroup at week 24 were 30.0% (placebo), 46.7% (100 mg), 57.7% (200 mg), and 69.6% (400/200 mg). E6011 administered for 24 weeks was well tolerated. Conclusion This is the first evidence that E6011, a novel cell trafficking inhibitor targeting the FKN-CX3 CR1 interaction, is modestly effective with 24 weeks of treatment in RA patients, although the primary end point was not met.

Fractalkine (CX3CL1/FKN) is a chemokine that regulates chemotaxis and adhesion of CX3C chemokine receptor 1 (CX3CR1)-expressing inflammatory cells. We conducted the first phase 1/2, open-label, multiple ascending dose study of E6011, a humanized anti-FKN monoclonal antibody, in Japanese rheumatoid arthritis (RA) patients (clinicaltrial.gov identifier: NCT02196558).Active RA patients with an inadequate response or intolerance to methotrexate or tumor necrosis factor (TNF) inhibitor received E6011 at week 0, 1, 2, and thereafter every 2 weeks for 12 weeks.Twelve, 15, and 10 subjects were enrolled in the 100, 200, and 400 mg cohorts, respectively. No severe adverse events (AEs) or deaths occurred, and no major differences were observed in the incidence or severity of AEs across the cohorts. Serum E6011 concentrations increased dose dependently. American College of Rheumatology (ACR) 20, 50, and 70 responses at week 12 were 75.0%, 33.3%, and 8.3% in the 100 mg cohort; 66.7%, 20.0%, and 13.3% in the 200 mg cohort; and 60.0%, 30.0%, and 20.0% in the 400 mg cohort, respectively.E6011 appeared to be safe and well tolerated in RA patients during this 12-week treatment period, suggesting that E6011 has an effective clinical response in active RA patients.

Background Fractalkine (CX3CL1, designated as FKN hereafter) is the sole member of the CX3C-chemokine which leads to dual actions, chemotaxis and cell adhesion for leukocytes expressing the cognate receptor, CX3CR1, during their migration. We have conducted clinical trials of E6011, a novel humanized anti-FKN monoclonal antibody, for patients with rheumatoid arthritis (RA) in Japan1. This is the first report of efficacy and safety results of E6011 from a Phase 2, multicenter, randomized, double-blind, placebo-controlled, parallel-group comparison study in RA patients inadequately responding to methotrexate (NCT02960438). Objectives To evaluate efficacy and safety of three dose of E6011 compared with placebo. Methods During the 24-week double-blind period, patients with moderately to severely active RA of inadequate response to MTX were randomly assigned to E6011 100 mg, 200 mg, 400/200 mg, or placebo groups at a 1:2:2:2 ratio. In the E6011 100 mg, 200 mg, and placebo groups, subjects received 100 mg, 200 mg, or placebo at Weeks 0, 1, 2, and every 2 weeks subsequently. In the E6011 400/200 mg group, subjects received 400 mg at Weeks 0, 1, 2, 4, 6, 8, 10 and then 200 mg every 2 weeks subsequently. Results A total of 190 subjects (54 in the placebo group, 28 in the 100 mg group, 54 in the 200 mg group, 54 in the 400/200 mg group) received study drug. Of the 190 subjects, 169 completed and 21 discontinued study treatment prematurely during the 24-week double-blind period. The ACR20 response rate at Week 12 (non-responder imputation), the primary endpoint, was 37.0% in the placebo group, 39.3% in the 100 mg group, 48.1% in the 200 mg group, and 46.3% in the 400/200 mg group (not statistically significant), and statistically significant difference from placebo in ACR20 response rate was found in the 200 mg and 400/200 mg groups at Week 24 (35.2% for placebo, 39.3% for 100 mg, 53.7% for 200 mg, 57.4% for 400/200 mg). In addition, we focused on CD16+ monocytes which highly expressing FKN receptor/CX3CR1 as a blood biomarker that linked to the clinical response to E6011. The whole patient population was divided into 2 groups according to the median value of baseline CD16+ monocyte percentage (Median: 10.35%). Much clearer ACR20 response was observed in a dose dependent manner in the subjects who showed higher baseline CD16+ monocytes over the median at Week 24 (NRI) (30.0% for placebo, 46.7% for 100 mg, 57.7% for 200 mg, and 69.6% for 400/200 mg) although such fashion was obscure in the subjects below the median value. Adverse events that occurred in ≥5% of subjects in any E6011 group were nasopharyngitis, upper respiratory tract infection, stomatitis, bronchitis, back pain, pharyngitis, and dental caries. As a results, E6011 was well tolerated with no notable safety concerns at doses of 100, 200, and 400/200 mg when administered subcutaneously for 24 weeks. Conclusion E6011 provided clinical improvements with a good safety profile in RA patients with inadequately responding to MTX. Especially, a higher efficacy of E6011 was suggested in patients with higher baseline CD16+ monocytes (%). This is the world-first evidence suggesting that a novel approach to target FKN/CX3CR1 interaction could be beneficial for RA. References [1] Tanaka Y, et al., Mod Rheumatol (2018) 28, 58-65 Acknowledgement The authors wish to thank the study investigators. Disclosure of Interests Yoshiya Tanaka Grant/research support from: Abbvie, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eisai, Mitsubishi-Tanabe, MSD, Ono, Taisho-Toyama, Takeda, Speakers bureau: Abbvie, Asahi-kasei, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eli Lilly, Eisai, Glaxo-Smithkline, Janssen, Mitsubishi-Tanabe, Novartis, Pfizer Japan Inc, Sanofi, Takeda, UCB, YL Biologics, Tsutomu Takeuchi Grant/research support from: Astellas Pharma Inc, Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Takeda Pharmaceutical Co., Ltd., AbbVie GK, Asahikasei Pharma Corp., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Eisai Co., Ltd., AYUMI Pharmaceutical Corporation, Nipponkayaku Co. Ltd., Novartis Pharma K.K., Grant/research support from: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Grant/research support from: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Abbivie GK, Nipponkayaku Co.Ltd, Janssen Pharmaceutical K.K., Astellas Pharma Inc., Taiho Pharmaceutical Co. Ltd., Chugai Pharmaceutical Co. Ltd., Taisho Toyama Pharmaceutical Co. Ltd., GlaxoSmithKline K.K., UCB Japan Co. Ltd., Consultant for: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Asahi Kasei Medical K.K., AbbVie GK, Daiichi Sankyo Co., Ltd., Bristol Myers Squibb, and Nipponkayaku Co. Ltd., Speakers bureau: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Speakers bureau: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Speakers bureau: AbbVie GK., Bristol–Myers K.K., Chugai Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Astellas Pharma Inc, Diaichi Sankyo Co. Ltd., Eisai Co. Ltd., Sanofi K.K., Teijin Pharma Ltd., Takeda Pharmaceutical Co. Ltd., Novartis Pharma K.K., Hisashi Yamanaka Grant/research support from: AbbVie, Eisai, Bristol-Meyers, Novartis, Behringer, Astellas, Kaken, Nippon-Shinyaku, Pfizer, UCB, Ayumi, Ono, Daiichi-Sankyo, Taisyo-Toyama, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, Torii, YLbio, Speakers bureau: Bristol-Meyers, Astellas, Pfizer, Daiichi-Sankyo, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, YLbio, Toshihiro Nanki Grant/research support from: Chugai, Eisai, Takeda, Teijin, Eli Lilly, Bristol-Myers, AbbVie, Ono, Novartis, Asahikasei, Mitsubishi-Tanabe, Astellas, Ayumi, Pfizer, Daiichi Sankyo, Shionogi, Sanofi, Nippon Kayaku, Yutoku, Actelion, UCB, Bayer, Nihon Pharmaceutical., Consultant for: UCB, Eisai, Chugai, Ono, Gilead., Speakers bureau: Mitsubishi-Tanabe, Chugai, Eisai, Takeda, Astellas, Janssen, Eli Lilly, Ayumi, Pfizer, Asahikasei, Sanofi, Daiichi Sankyo, Otsuka, AbbVie, Ono, Teijin, Nippon Kayaku, UCB., Hisanori Umehara: None declared, Nobuyuki Yasuda Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Fumitoshi Tago Employee of: Eisai Co., Ltd., Yasumi Kitahara Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Makoto Kawakubo Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Kentaro Torii Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Seiichiro Hojo Employee of: Eisai Co., Ltd., Tetsu Kawano Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Toshio Imai Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.)

Background Fractalkine (CX3CL1, designated as FKN hereafter) is the sole member of the CX3C-chemokine which leads to dual actions, chemotaxis and cell adhesion for leukocytes expressing the cognate receptor, CX3CR1, during their migration. We have conducted clinical trials of E6011, a novel humanized anti-FKN monoclonal antibody, for patients with rheumatoid arthritis (RA) in Japan1. This is the first report of a Phase 2, multicenter, randomized, double-blind, placebo-controlled, parallel-group comparison study of E6011 in RA patients inadequately responding to biologics (NCT02960490). Objectives To evaluate efficacy and safety of E6011 compared with placebo. Methods During the 24-week double-blind period, patients with moderately to severely active RA of inadequate response to biologics were randomly assigned to E6011 400 mg or placebo groups at a 1:1 ratio. Patients who continued the study beyond Week 12 were further allocated to E6011 200 mg or 400 mg at a 1:1 ratio within the initially assigned group. Patients received either E6011 400 mg or placebo at Weeks 0, 1, 2, and every 2 weeks subsequently until Week 10 and then E6011 200 mg or 400 mg every 2 weeks between Weeks 12 and 22 in a double-blind manner. This abstract reports the results from the first 12-week placebo controlled period. Results A total of 64 subjects (33 in the placebo group and 31 in the E6011 400 mg group) received study drug. Of the 64 subjects, 55 completed and 9 discontinued study treatment prematurely during the 12-week placebo controlled double-blind period. The ACR20 response rate at Week 12 (non-responder imputation), the primary endpoint, was 27.3% (9/33 subjects) in the placebo group and 22.6% (7/31 subjects) in the E6011 group. ACR50 and ACR70 response rate at Week 12 were 3.0%, 0% in the placebo group and 9.7%, 3.2% in the E6011 group, respectively. Numerically greater improvement from baseline was found for some parameters of the ACR components in the E6011 group, however, no statistically significant differences were found in any of the ACR components between the placebo and E6011 groups. From the exploratory PK exposure analysis, there was a tendency that the effect of E6011 became clearer in the subjects who achieved higher serum trough E6011 concentration. Adverse events that occurred in at least 2 subjects in the E6011 group were stomatitis, injection site erythema, nasopharyngitis, and blood creatine phosphokinase increased. As a results, E6011 was well tolerated with no notable safety concerns at doses of 400 mg when administered subcutaneously for 12 weeks. Conclusion E6011 400 mg was well tolerated but did not show clear efficacy at Week 12 compared with placebo in RA patients with inadequately responding to biologics. Further investigation to seek an optimal clinical dose and evaluation period of E6011 are warranted. Reference [1] Tanaka Y, et al., Mod Rheumatol (2018) 28, 58-65 Acknowledgement The authors wish to thank the study investigators. Disclosure of Interests Yoshiya Tanaka Grant/research support from: Abbvie, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eisai, Mitsubishi-Tanabe, MSD, Ono, Taisho-Toyama, Takeda, Speakers bureau: Abbvie, Asahi-kasei, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eli Lilly, Eisai, Glaxo-Smithkline, Janssen, Mitsubishi-Tanabe, Novartis, Pfizer Japan Inc, Sanofi, Takeda, UCB, YL Biologics, Tsutomu Takeuchi Grant/research support from: Astellas Pharma Inc, Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Takeda Pharmaceutical Co., Ltd., AbbVie GK, Asahikasei Pharma Corp., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Eisai Co., Ltd., AYUMI Pharmaceutical Corporation, Nipponkayaku Co. Ltd., Novartis Pharma K.K., Grant/research support from: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Grant/research support from: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Abbivie GK, Nipponkayaku Co.Ltd, Janssen Pharmaceutical K.K., Astellas Pharma Inc., Taiho Pharmaceutical Co. Ltd., Chugai Pharmaceutical Co. Ltd., Taisho Toyama Pharmaceutical Co. Ltd., GlaxoSmithKline K.K., UCB Japan Co. Ltd., Consultant for: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Asahi Kasei Medical K.K., AbbVie GK, Daiichi Sankyo Co., Ltd., Bristol Myers Squibb, and Nipponkayaku Co. Ltd., Speakers bureau: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Speakers bureau: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Speakers bureau: AbbVie GK., Bristol–Myers K.K., Chugai Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Astellas Pharma Inc, Diaichi Sankyo Co. Ltd., Eisai Co. Ltd., Sanofi K.K., Teijin Pharma Ltd., Takeda Pharmaceutical Co. Ltd., Novartis Pharma K.K., Hisashi Yamanaka Grant/research support from: AbbVie, Eisai, Bristol-Meyers, Novartis, Behringer, Astellas, Kaken, Nippon-Shinyaku, Pfizer, UCB, Ayumi, Ono, Daiichi-Sankyo, Taisyo-Toyama, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, Torii, YLbio, Speakers bureau: Bristol-Meyers, Astellas, Pfizer, Daiichi-Sankyo, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, YLbio, Toshihiro Nanki Grant/research support from: Chugai, Eisai, Takeda, Teijin, Eli Lilly, Bristol-Myers, AbbVie, Ono, Novartis, Asahikasei, Mitsubishi-Tanabe, Astellas, Ayumi, Pfizer, Daiichi Sankyo, Shionogi, Sanofi, Nippon Kayaku, Yutoku, Actelion, UCB, Bayer, Nihon Pharmaceutical., Consultant for: UCB, Eisai, Chugai, Ono, Gilead., Speakers bureau: Mitsubishi-Tanabe, Chugai, Eisai, Takeda, Astellas, Janssen, Eli Lilly, Ayumi, Pfizer, Asahikasei, Sanofi, Daiichi Sankyo, Otsuka, AbbVie, Ono, Teijin, Nippon Kayaku, UCB., Hisanori Umehara: None declared, Nobuyuki Yasuda Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Fumitoshi Tago Employee of: Eisai Co., Ltd., Yasumi Kitahara Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Makoto Kawakubo Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Hayato Hisaki Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Seiichiro Hojo Employee of: Eisai Co., Ltd., Tetsu Kawano Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Toshio Imai Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.)

Hepatocyte growth factor (HGF) is an endogenously expressed bioactive substance that has a strong anti-apoptotic effect. In this study, we biochemically and histologically characterized the effects of rh-HGF on in vitro human hepatocyte injury and mouse acute liver failure (ALF) models, both of which were induced by antibody-mediated Fas signaling. rh-HGF inhibited intracellular caspase-3/7 activation and cytokeratin 18 (CK-18) fragment release in both models. Histologically, rh-HGF dramatically suppressed parenchymal damage and intrahepatic hemorrhage. Among the laboratory parameters, prothrombin time (PT) was strongly preserved by rh-HGF, and PT was well correlated with the degree of intrahepatic hemorrhage. These results showed that the anti-apoptotic effect of rh-HGF on hepatocytes coincided strikingly with the suppression of intrahepatic hemorrhage. PT was considered to be the best parameter that correlated with the intrahepatic hemorrhages associated with hepatocellular damage. The action of rh-HGF might derive not only from its anti-apoptosis effects on liver parenchymal cells but also from its stabilization of structural and vasculature integrity.

To assess the preclinical efficacy and mechanism of action of an anti-CXCultured human dermal fibroblasts were used to evaluate the direct effect of anti-CXAnti-CXAnti-CX

Background:Fractalkine (FKN) and its solo receptor CX3CR1 are deeply involved in the pathogenesis of rheumatoid arthritis (RA). FKN is expressed on vascular endothelium, while CX3CR1 is expressed on peripheral blood leukocytes such as monocytes, macrophages, NK cells, effector CD8+T cells and a minor fraction of CD4+T cells. E6011, a novel humanized anti-FKN monoclonal antibody (mAb), is under clinical development in RA.Objectives:In order to continuously assess the E6011 pharmacodynamics by monitoring the alteration of peripheral blood immune cells, including CX3CR1-expressed cell populations, a series of multi-color flow cytometry (FCM) was conducted before and during the course of the E6011 treatment of active RA patients in phase 2 clinical trial.Methods:Immuno-phenotypic changes were explored by FCM during the E6011 administration in 190 Japanese RA patients with inadequate response to MTX (NCT02960438). Patient’s peripheral blood were drawn into fixative tube (Cyto-Chex® BCT, Streck) at each clinics and thereafter transported to the FCM facility at KAN Research Institute, Inc. within 30 hours after the blood collection to operate the FCM analysis by standardized method. Immuno-phenotyping was carried out by multi colors flow cytometry (BD FACSCantoIITM, BD LSRFortessaTM, BD Biosciences).Results:Based on these determined conditions, CX3CR1 expression on monocytes, NK cells and a part of CD8+and CD4+T cells were confirmed in this method. Interestingly, during the E6011 treatment, the proportion of CD16+monocytes, which highly express CX3CR1 within whole monocytes, were significantly decreased at 2 week after initial treatment from the baseline (E6011:p< 0.001, placebo:p> 0.48) and sustained up to 24 week, while that of CD16-monocytes were increased. The reduction of the frequency of CX3CR1+cells in NK cells, CD4+and CD8+T cells were not observed, but in some certain populations like CX3CR1-expressed CD56+CD16+NK cells and terminal differentiated effector CD8+T cells, the frequency of CX3CR1+cells in these populations tended to increase from the baseline at 2 week and kept increasing up to 24 week by the E6011 treatment.Conclusion:E6011 significantly decreased the proportion of CD16+monocytes in whole monocytes. Our results indicated that the reduction of CD16+monocytes after initial treatment might be a sensitive marker of E6011 in peripheral blood, possibly reflecting mechanism of action of E6011, since the CD16+monocytes highly express CX3CR1.References:[1]Tanaka Y, et al., Mod Rheumatol (2018) 28, 58-65Disclosure of Interests:Tomohiro Yamada Employee of: KAN Research Institute, Inc,, Jungo Kakuta Employee of: KAN Research Institute, Inc., Eri Fusaoka-Nishioka Employee of: KAN Research Institute, Inc., Jun-ichi Ito Employee of: EISAI, Nobuyuki Yasuda Employee of: KAN Research Institute, Inc., Tetsu Kawano: None declared, Toshio Imai: None declared

E6011 is a novel humanized antifractalkine (FKN) monoclonal antibody being developed as a therapeutic target for Crohn's disease, rheumatoid arthritis, and primary biliary cholangitis. This study was a randomized, double-blind, placebo-controlled single-ascending-dose study of intravenous administration of E6011 (0.0006-10 mg/kg) in healthy Japanese adult men (n = 64). The starting dose was the minimum anticipated biological effect level (MABEL). MABEL was estimated by extrapolating results of a pharmacokinetic/pharmacodynamic (PK/PD) model relating E6011 exposure and suppression of free soluble FKN using data obtained from cynomolgus monkeys. Safety assessments consisted of monitoring and recording adverse events, laboratory tests, vital signs, intensive electrocardiograms, and chest x-rays. Blood samples to determine PK, PD (serum total FKN concentration), and serum anti-E6011 antibody were collected. Noncompartmental analysis was used to derive PK parameters. Single intravenous infusions of E6011 were safe and well tolerated in healthy subjects. Serum E6011 concentrations showed triphasic elimination. An increase in serum total FKN concentration was observed, confirming target engagement. The dose strategy for patient studies is to select regimens that will attain a minimum serum E6011 exposure of 10 μg/mL, identified as the minimum concentration needed to saturate the target-mediated elimination pathway. Model-based drug development from preclinical stage was successful in identifying dose regimens for clinical testing.

Acute liver failure is a potentially fatal disease of various etiologies for which liver transplantation is the only known curative treatment. Although the decision-making on transplantation is largely dependent on the severity of liver injury (based on predicting a fatal outcome), a statistical analysis to predict “survival” has not been extensively conducted. In this study, we investigate the medical history of patients in two distinct areas of Japan with the aim of identifying the predictors of survival in patients with acute liver injury (ALI). Datasets of 301 patients with ALI in two distinct areas (93 in southern Kyushu and 208 in northern Tohoku) of Japan, who were treated from 2004 to 2014, were included in the analysis. Among the enrolled 301 cases, 263 patients survived without transplantation. A PT-INR of ≥ 1.3 during the clinical course was found to be adequate for predicting a poor prognosis, because all of the fatal cases emerged from this population (hazard ratios: southern Kyushu, 0.2827; northern Tohoku, 0.1862). All surviving patients showed a reduction in their PT-INR during treatment, whereas the PT-INR did not decrease in the patients with a poor prognosis. A PT-INR of

NKT cells, a novel murine lymphoid lineage bearing an invariant T cell receptor encoded by V alpha 14 and J alpha 281 gene segments, recognize a specific ligand glycolipid, alpha-galactosylceramide (alpha-GalCer) in a CD1d-dependent manner. Recent research has revealed that activated V alpha 14 NKT cells have dramatic antitumor effects against a wide variety of tumor cell lines in vivo and in vitro. Here, we demonstrate strong in vivo antitumor effects brought about by treatment with alpha-GalCer-pulsed dendritic cells in comparison with in vitro-activated V alpha 14 NKT cells. Furthermore, we show a significant expansion of endogenous V alpha 14 NKT cells in the lung following the administration of alpha-GalCer-pulsed dendritic cells. The feasibility of immunotherapy with alpha-GalCer-pulsed dendritic cells is discussed.

Experimental autoimmune encephalomyelitis (EAE) is a Th1 cell-mediated autoimmune disease that can be protected against by stimulating regulatory cells. Here we examined whether EAE can be purposefully modulated by stimulating Vα14 NK T cells with the CD1d-restricted ligand α-galactosylceramide (α-GC). EAE induced in wild-type C57BL/6 (B6) mice was not appreciably altered by injection of α-GC. However, EAE induced in IL-4 knockout mice and IFN-γ knockout mice was enhanced or suppressed by α-GC, respectively. This indicates that the IL-4 and IFN-γ triggered by α-GC may play an inhibitory or enhancing role in the regulation of EAE. We next studied whether NK T cells of wild-type mice may switch their Th0-like phenotype toward Th1 or Th2. Notably, in the presence of blocking B7.2 (CD86) mAb, α-GC stimulation could bias the cytokine profile of NK T cells toward Th2, whereas presentation of α-GC by CD40-activated APC induced a Th1 shift of NK T cells. Furthermore, transfer of the α-GC-pulsed APC preparations suppressed or enhanced EAE according to their ability to polarize NK T cells toward Th2 or Th1 in vitro. These results have important implications for understanding the role of NK T cells in autoimmunity and for designing a therapeutic strategy targeting NK T cells.

In vivo administration of NKT cell ligand, alpha-galactosylceramide (alpha-GalCer), caused the activation of NKT cells to induce a strong NK activity and cytokine production by CD1d-restricted mechanisms. Surprisingly, we also found that alpha-GalCer induced the activation of immunoregulatory cells involved in acquired immunity. Specifically, in vivo administration of alpha-GalCer resulted in the induction of the early activation marker CD69 on CD4(+) T cells, CD8(+) T cells and B cells in addition to macrophages and NKT cells. However, no significant induction of CD69 was observed on cells from CD1d- or V(alpha)14 NKT-deficient mice, indicating an essential role for the interaction between NKT cells and CD1d-expressing dendritic cells (DC) in the activation of acquired immunity in response to alpha-GalCer. Indeed, in vivo injection of alpha-GalCer resulted not only in the activation of NKT cells but also in the generation of CD69(+)CD8(+) T cells possessing both cytotoxic T lymphocyte (CTL) activity and IFN-gamma-producing ability. Tumor-specific CTL generation was also accelerated by alpha-GalCer. The critical role of CD40-CD40 ligand (CD40L)-mediated NKT-DC interaction during the development of CD69(+)CD8(+) CTL by alpha-GalCer was demonstrated by blocking experiments using anti-CD40L mAb. These findings provide direct evidence for a critical role of CD1d-restricted NKT cells and DC in bridging innate and acquired immunity.

The administration of concanavalin A (Con A) induces a rapid severe injury of hepatocytes in mice. Although the Con A-induced hepatitis is considered to be an experimental model of human autoimmune hepatitis, the precise cellular and molecular mechanisms that induce hepatocyte injury remain unclear. Here, we demonstrate that Valpha14 NKT cells are required and sufficient for induction of this hepatitis. Moreover, interleukin (IL)-4 produced by Con A-activated Valpha14 NKT cells is found to play a crucial role in disease development by augmenting the cytotoxic activity of Valpha14 NKT cells in an autocrine fashion. Indeed, short-term treatment with IL-4 induces an increase in the expression of granzyme B and Fas ligand (L) in Valpha14 NKT cells. Moreover, Valpha14 NKT cells from either perforin knock-out mice or FasL-mutant gld/gld mice fail to induce hepatitis, and hence perforin-granzyme B and FasL appear to be effector molecules in Con A-induced Valpha14 NKT cell-mediated hepatocyte injury.

alpha-Galactosylceramide (alpha-GalCer), a glycolipid antigen, specifically activates natural killer T (NKT) cells by a CD1d-restricted mechanism. In this work, we found that in vivo administration of alpha-GalCer resulted in the activation of B cells in addition to NKT cells, namely, alpha-GalCer administration caused upregulation of the early activation marker, CD69, on both NKT and B cells. In addition, expression of B7.2 and I-A(b) on B cells was greatly upregulated by alpha-GalCer. However, serum levels of IgE, IgG1, and IgG2a were not significantly changed within 48 h. In the present experiments, it was also demonstrated that the upregulation of CD69 expression by alpha-GalCer was strongly blocked by anti-IL-4 monoclonal antibody. Moreover, B-cell activation by alpha-GalCer was not observed in NKT-deficient mice. These results suggested that antigen-stimulated NKT cells might play a critical role not only in early defense mechanisms but also in early B-cell activation through IL-4 production.

The combined therapeutic effect of natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) and IL-12 against highly metastatic B16-BL6-HM melanoma cells was investigated. In comparison with a single administration of alpha-GalCer or IL-12, the combined treatment of tumor-bearing mice with alpha-GalCer plus IL-12 caused a super-induction of serum IFN-gamma levels, though alpha-GalCer-induced IL-4 production was rather inhibited. In parallel with the augmented IFN-gamma production, the natural killing activity against YAC-1 cells and syngeneic B16-BL6-HM melanoma was greatly augmented by the combined therapy. The major effector cells responsible for natural killing activity induced by alpha-GalCer plus IL-12 were enriched in both NK1.1+ TCRalphabeta+ NKT cells and NK1.1+ TCRalphabeta- NK cells. The preventing effect of alpha-GalCer or IL-12 alone against lung metastasis of B16-BL6-HM was also enhanced by the combination therapy. The antitumor activity of alpha-GalCer was totally abolished in NKT-deficient mice. However, IL-12-induced antitumor activity was not eliminated in NKT-deficient mice though it was inhibited by anti-asialo GM1 Ab treatment. These findings suggested that alpha-GalCer synergistically act with IL-12 to activate both NKT cells and NK cells, which may play a critical role in the strong prevention of distant tumor metastasis at early stages of tumor-bearing. These data will provide a novel tool for the prevention of tumor metastasis using NKT-specific ligands alpha-GalCer and IL-12.

A unique lymphoid lineage, Vα14 NKT cells, bearing an invariant Ag receptor encoded by Vα14 and Jα281 gene segments, play crucial roles in various immune responses, including protective immunity against malignant tumors. A specific ligand of Vα14 NKT cells is determined to be α-galactosylceramide (α-GalCer) which is presented by the CD1d molecule. Here, we report that dendritic cells (DCs) pulsed with α-GalCer effectively induce potent antitumor cytotoxic activity by specific activation of Vα14 NKT cells, resulting in the inhibition of tumor metastasis in vivo. Moreover, a complete inhibition of B16 melanoma metastasis in the liver was observed when α-GalCer-pulsed DCs were injected even 7 days after transfer of tumor cells to syngeneic mice where small but multiple metastatic nodules were already formed. The potential utility of DCs pulsed with α-GalCer for tumor immunotherapy is discussed.

NK1.1+αβ T cells emerge in the peritoneal cavity after an i.p. infection with Salmonella choleraesuis in mice. To elucidate the role of the NK1.1+αβ T cells during murine salmonellosis, mice lacking NK1.1+αβ T cells by disruption of TCRβ (TCRβ−/−), β2m (β2m−/−), or Jα281 (Jα281−/−) gene were i.p. inoculated with S. choleraesuis. The peritoneal exudate T cells in wild type (wt) mice on day 3 after infection produced IL-4 upon TCRαβ stimulation, whereas those in TCRβ−/−, β2m−/−, or Jα281−/− mice showed no IL-4 production upon the stimulation, indicating that NK1.1+αβ T cells are the main source of IL-4 production at the early phase of Salmonella infection. Neutralization of endogenous IL-4 by administration of anti-IL-4 mAb to wt mice reduced the number of Salmonella accompanied by increased IL-12 production by macrophages after Salmonella infection. The IL-12 production by the peritoneal macrophages was significantly augmented in mice lacking NK1.1+αβ T cells after Salmonella infection accompanied by increased serum IFN-γ level. The aberrantly increased IL-12 production in infected TCRβ−/− or Jα281−/− mice was suppressed by adoptive transfer of T cells containing NK1.1+αβ T cells but not by the transfer of T cells depleted of NK1.1+αβ T cells or T cells from Jα281−/− mice. Taken together, it is suggested that NK1.1+αβ T cells eliciting IL-4 have a regulatory function in the IL-12 production by macrophages at the early phase of Salmonella infection.

The balance between Th1 and Th2 development is determined by IL-4 and IL-12. While the role for CD4+ NK1.1+ T (NKT) cells in influencing this balance has been recognized based on their capacity to produce IL-4, it is unknown how IL-12 is produced in the innate immune system in which they participate. This study demonstrates that Ag-activated CD4+ NKT cells express CD40 ligand (CD40L) (CD154), which engages CD40 on APC and stimulates them to produce IL-12. Culture of B cell-depleted spleen cells from C57BL/6 mice with α-galactosylceramide (α-GalCer) capable of selectively stimulating Vα14/Jα281+ NKT cells resulted in the production of IL-12 together with IFN-γ and IL-4. α-GalCer-induced IL-12 production occurred in I-Abβ-deficient mice, but not in β2-microglobulin-deficient and Vα14/Jα281 TCR-deficient mice, and was inhibited by anti-CD40L mAb. Of CD4+ and CD4− NKT cells, the capacity to express CD40L/CD154 and trigger IL-12 production following α-GalCer stimulation was exhibited preferentially by the CD4+ NKT subset. IL-12 production was also observed in α-GalCer-treated mice. Production of IL-12 preceded IFN-γ production, and IL-12 was required for IFN-γ, but not IL-4, production. A stimulatory/inhibitory relationship existed between IL-12 and IL-4 production. These results illustrate a novel function of CD4+ NKT cells that could be involved in the regulation of Th1 vs Th2 development.

Receptors for bioactive glycosylation-inhibiting factor (GIF) were demonstrated using a bioactive mutant of recombinant human (rh) GIF, which is comparable to the suppressor T (Ts) cell-derived bioactive GIF in its affinity for the receptors on helper T (Th) hybridoma cells. Both naive T and B cells in normal mouse spleen lacked GIF receptors. However, presentation of specific antigen to naive T cells resulted in the expression of the receptors on activated T cells. Furthermore, activation of small resting B cells with F(ab')2 fragments of anti-mouse IgM plus IL-4, lipopolysaccharide (LPS) plus IL-4 or LPS plus dextran sulfate induced the expression of the receptors within 48 h of B cell stimulation. It was also found that NK T cells freshly isolated from mouse spleen, but not conventional NK cells, expressed receptors for GIF. CD4(+) and CD4(-) subpopulations of NK T cells showed a similar binding capability. Mature dendritic cells derived from bone marrow did not bear the receptors. The dissociation constant (Kd) of the interaction between the bioactive rhGIF mutant and the high-affinity receptors was 10-100 pM, whereas inactive wild-type rhGIF failed to bind to the receptors. A bioactive derivative of rhGIF suppressed both IgG1 and IgE synthesis by purified B cells activated by LPS and IL-4, indicating that the binding of bioactive GIF to its receptors on activated B cells results in suppression of their differentiation.

Murine NKT cells can recognize alpha-galactosylceramide (alpha-GalCer) in the context of a class Ib CD1d molecule. Here we show that alpha-GalCer can selectively activate freshly isolated human Valpha24(+)Vbeta11(+) cells, functionally defining the human NKT cells. The naive human NKT cell repertoire consisted of cells expressing an invariant Valpha24JalphaQ chain and a diverse array of beta chains derived from a single Vbeta11 gene segment. Stimulation with alpha-GalCer expanded a polyclonal subset of the human NKT cell repertoire carrying a novel complementarity-determining region (CDR) 3beta consensus motif that may directly interact with the sugar moiety of alpha-GalCer. Our data suggest that certain redundancy is allowed for CDR3beta of NKT antigen receptor to interact with the ligand and provide a first clue to understand the novel protein-carbohydrate interaction mechanisms.

Murine Valpha14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Valpha14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-gamma upon in vivo stimulation with a specific ligand, alpha-galactosylceramide (alpha-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Valpha14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Valpha14 NKT-deficient mice that Valpha14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with alpha-GalCer, Valpha14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Valpha14 NKT- or IFN-gamma-deficient mice. Consistent with these results, we also found that ligand-activated Valpha14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Valpha14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, our studies have revealed that IL-4 produced by Valpha14 NKT cells has only a minor effect on Th2 cell differentiation.

A novel lymphoid lineage, NK T cells, was recently found. The NK T cells are the major population in the periphery comprising 5% of splenic T cells and 40% of bone marrow T cells. They express a unique TCR composed of invariant Vα14Jα281 and Vβ8.2 together with NK receptor (NKRPI). Surprisingly, the invariant Vα14 + TCR is exclusively expressed on NK T cells but not on conventional T cells. As the selective decrease in Vα14 + NK T cell population in the periphery is tightly correlated with autoimmune disease development, Vα14 + NK T cells control development of autoimmune diseases. We also found that Vα14 TCR gene rearrangement and transcripts were detected at an early embryogenesis (d9.5) before the thymus formation. Therefore NK T cells are in the distinct category from conventional T cells. The target of NK T cells is found to be CD1 (class 1b, monomorphic class I MHC-like molecule) present on bone marrow–derived cells and is killed by Fas-FasL interaction or perforin-mediated mechanisms. These results indicate that NK T cells consist of an immunoregulatory system different from defense system in terms of homogeneous repertoire, extrathymic development in early stage of gestation, and their regulatory functional role. (J Allergy Clin Immunol 1996;98:S263-9)

Background Fractalkine (CX3CL1, designated as FKN hereafter) is the sole member of the CX3C-chemokine which leads to dual actions, chemotaxis and cell adhesion for leukocytes expressing the cognate receptor, CX3CR1, during their migration. Accumulating evidence is telling that FKN-CX3CR1 axis plays a pivotal role in leukocyte/lymphocyte accumulation in inflamed tissues in RA1. We developed E6011, a novel humanized anti-FKN monoclonal antibody. Objectives To evaluate safety, pharmacokinetics and efficacy of E6011 in a Phase 1/2, open-label, multiple ascending dose study in RA patients for the first time (NCT02196558). Methods Active RA patients with inadequate response to MTX or TNF inhibitors were received 7 consecutive doses (subcutaneous: SC) of E6011 at week 0, 1, 2 and thereafter every 2 weeks up to week 10. A hundred and 200 mg of E6011 were chosen for the study as putatively appropriate doses for patients by the PK/PD modeling previously obtained in the Ph1 healthy volunteer study of E6011. Results Twelve subjects were enrolled in the cohort of 100 mg and subsequently 15 subjects were enrolled in the 200 mg cohort, in total, 27 subjects received repeated subcutaneous administrations of E6011. As a result, repeated dose of E6011 was found safe and well tolerated. The incidence of AE, treatment-related AE and SAE was 59.3%, 29.6% and 7.4%, respectively. There were no severe AE or deaths, and no significant differences were observed in the incidence or severity of AE between the 100 mg and 200 mg cohorts. After starting multiple SC injection of E6011, serum E6011 concentration reached steady-state at week 2, and its level was maintained up to week 12. Mean trough E6011 concentrations from week 2 through week 12 were 11.6 – 15.7 ug/mL and 29.2 – 38.8 ug/mL after dosing of 100 and 200 mg, respectively. Clinical outcome was also available in the study in which response rates of ACR20, 50 and 70 at week 12 were 75.0%, 33.3%, 8.3% and 80.0%, 26.7%, 20.0% in the cohort of 100 and 200 mg, respectively. At week 12, 33.3% of subjects achieved DAS28-CRP remission in both cohorts, 16.7% and 20.0% for SDAI remission and 8.3% and 26.7% for Boolean remission were observed in the 100 and 200 mg cohorts, respectively. Conclusions E6011 was safe and well tolerated, and demonstrated a promising efficacy in active RA patients with MTX or TNFi-IR. While further clinical studies are required, the results obtained indicate that a novel biological DMARD targeting FKN/CX3CR1 interaction will be clinically beneficial for active RA patients. References Nanki T. Arthritis Rheum. 2002; 46(11):2878–83. Acknowledgement The authors wish to thank the study investigators. Disclosure of Interest Y. Tanaka Grant/research support from: Mitsubishi-Tanabe, Takeda, Chugai, Astellas, Eisai, Taisho-Toyama, Kyowa-Kirin, AbbVie, Bristol-Myers, Speakers bureau: AbbVie, Daiichi-Sankyo, Chugai, Takeda, Mitsubishi-Tanabe, Bristol-Myers, Astellas, Eisai, Janssen, Pfizer, Asahi-kasei, Eli Lilly, GlaxoSmithKline, UCB, Teijin, MSD, Santen, T. Takeuchi Grant/research support from: Astellas, Bristol-Myers, Chugai, Daiichi-Sankyo, Eisai, AYUMI, Takeda, Teijin, AbbVie, Asahikasei, Taisho-Toyama, Consultant for: Astra Zeneca, Eli Lilly, Novartis, Mitsubishi-Tanabe, AsahiKasei, AbbVie, Daiichi-Sankyo, Bristol-Myers, Nipponkayaku, Janssen, Merck Serono, Takeda, Astellas, Speakers bureau: AbbVie, Bristol-Myers, Chugai, Eisai, Janssen, Mitsubishi-Tanabe, Pfizer, Takeda, Astellas, Daiichi-Sankyo, Celtrion, Nipponkayaku, H. Umehara: None declared, T. Nanki Grant/research support from: Chugai, Eisai, Takeda, Teijin, Eli Lilly, Bristol-Myers, AbbVie, Mitsubishi-Tanabe, Ono, Daiichi-Sankyo, Shionogi, Asahikasei, Nippon Boehringer Ingelheim, Ajinomoto, Consultant for: UCB, Eisai, Chugai, Speakers bureau: Mitsubishi-Tanabe, Chugai, Eisai, Takeda, Astellas, Janssen, Eli Lilly, Ono, AbbVie, Bristol-Myers, Daiichi-Sankyo, Santen, H. Akama Shareholder of: EISAI, Employee of: EISAI, N. Yasuda Shareholder of: EISAI, Employee of: EISAI, F. Tago Employee of: EISAI, M. Kawakubo Shareholder of: EISAI, Employee of: EISAI, S. Hojo Employee of: EISAI, T. Kawano Employee of: KAN Research Institute, T. Imai Employee of: KAN Research Institute

Background CC chemokine ligand 20 (CCL20) and CC chemokine receptor 6 are believed to stimulate the recruitment of neutrophils and activation of macrophages against bacterial pathogens through the activation of T helper cells. We analyzed the role of CCL20 in the acute phase of sepsis. Methods The effect of a neutralizing, anti-mouse CCL20 monoclonal antibody (mAb) was examined in 2 murine models of sepsis: Cecal ligation and puncture (CLP) and Escherichia coli peritonitis. Immune cell migration, bacterial clearance, and expression of 17 cytokines and 5 chemokines were quantified in E coli-induced peritonitis. Expression of CCL20 in various tissues was determined, and apoptotic cells in jejunum were measured. Results Anti-CCL20 mAb increased mortality in CLP and E coli peritonitis (P = .029 and .024, respectively by Kaplan–Meier method and log-rank test). The 48-hour survival rate in anti-CCL20 mAb- and control immunoglobulin (Ig)G-treated mice was 37% (11/30) vs 62% (18/29) in CLP and 28% (11/40) vs 48% (19/40) in bacterial peritonitis. Neutralization of CCL20 showed no effect on leukocyte infiltration into the peritoneal cavity or bacterial clearance at 24 hours. CCL20 was induced strongly and predominantly in jejunum after bacterial infection, and neutralizing CCL20 increased apoptosis of epithelial cells in jejunum crypt. Inhibition of CCL20 increased serum tumor necrosis factor (TNF)-α (3.3-fold greater than control mice) and decreased serum interleukin (IL)-1α and IL-6. Conclusion Neutralization of CCL20 before induction of sepsis increased mortality during sepsis accompanied with increasing epithelial apoptosis in the jejunum and augmenting serum TNF-α.

The inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) are illness characterized by a chronic clinical course of relapse and remission associated with self-destructive inflammation of the gastrointestinal tract. In both UC and CD, leukocyte infiltration into the intestine is fundamental event in disease development and progression where the chemokines and their receptors are orchestrating the tissue-specific and the cell type-selective trafficking of leukocytes. In this review, we will discuss the homeostatic and inflammatory roles of the chemokines and their receptors with their potentials and promise as molecular targets for therapeutic interventions in human IBD, focusing on the recently identified role of the CX3CL1-CX3CR1 axis, as well as the CCL20-CCR6, CCL25-CCR9, and CXCL10-CXCR3 pathways.

A novel lymphocyte, NKT cells bearing an invariant V(alpha)14 antigen receptor, specifically recognizes alpha-galactosylceramide (alpha-GalCer) exclusively presented by mouse CD1d (mCD1d). However, the precise molecular interaction remains unclear. For the basis of functional analyses, a docking model of alpha-GalCer with the crystal structure of mCD1d was constructed. Possible residues involved in the alpha-GalCer--mCD1d interaction were found to be Arg79, Glu83 and Asp80 for carbohydrate recognition, and Asp153 for interaction with the amide group on the fatty acyl chain. The alpha-GalCer-presenting ability of various transfectants expressing mutant mCD1d was completely abrogated if a single amino acid mutation was induced at positions 79, 80, 83 or 153, suggesting that the polar amino acids above the F' pocket are crucial for alpha-GalCer presentation to activate V(alpha)14 NKT cells. The possibility that Glu83 is a contact site for the NKT cell receptor is also discussed.

Valpha14 natural killer T (NKT) cells produce large amounts of both IL-4 and IFN-gamma upon stimulation with a ligand, alpha-galactosylceramide (alpha-GalCer), and play a crucial role in various immune responses, including allergic diseases. Interestingly, Valpha14 NKT cells are not essential for the induction of IgE responses but rather induce suppression of specific IgE production upon activation. The suppression in the IgE production is not detected either in Valpha14 NKT cell-deficient mice or in IFN-gamma-deficient mice. Thus, activated Valpha14 NKT cells are likely to exert a potent suppressive activity on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, little regulatory effect of IL-4 produced by Valpha14 NKT cells on Th2 cell differentiation is suggested.

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset–depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1+ T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3− NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I–deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jα281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or α-galactosylceramide.

The immunological mechanisms that regulate abortion are largely unknown. Here, we found that a distinct subset of lymphocytes, Valpha14 NKT cells expressing an invariant antigen receptor encoded by Valpha14/Jalpha281 and Vbeta7 segments, accumulated in the decidua during pregnancy and provoked abortion upon stimulation with alpha-galactosylceramide (alpha-GalCer), a specific ligand for Valpha14 NKT cells. The alpha-GalCer-mediated abortion was not observed in Valpha14 NKT-, IFN-gamma-, tumor necrosis factor alpha-, or perforin-knock-out mice and appeared to be due to the degeneration of embryonic trophoblasts mediated by the activated Valpha14 NKT cells whose perforin-dependent killing and production of IFN-gamma and tumor necrosis factor alpha were essential. The possible role of the decidual Valpha14 NKT cells in the pathogenesis of abortion is discussed.

The role of natural killer T (NKT) cells in the prevention of multiple tumor metastasis was examined. The i.v. inoculation of a highly metastatic subline of B16-BL6 (B16-BL6-HM) melanoma cells resulted in the formation of metastatic nodules in lymph nodes in addition to lung, intrapleural cavity, and ovary. However, treatment of the mice with the NKT cell ligand alpha-galactosylceramide (alpha-GalCer) three times from 1 day after B16-BL6-HM melanoma inoculation caused a significant inhibition of multiple metastasis. Lymph node metastasis of B16-BL6-HM was almost completely blocked by alpha-GalCer treatment. This antimetastatic effect of alpha-GalCer was abolished in NKT cell-deficient mice. These results suggest that alpha-GalCer-activated NKT cells played a critical role in the prevention of lymph node metastasis of melanoma cells.

The natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12-mediated antitumor activities. Because of these similarities between the activities of alpha-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by alpha-GalCer. We first established, using purified subsets of various lymphocyte populations, that alpha-GalCer selectively activates NKT cells for production of interferon (IFN)-gamma. Production of IFN-gamma by NKT cells in response to alpha-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, alpha-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1(-/-) or Valpha14(-/-) mice. This effect of alpha-GalCer required the production of IFN-gamma by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of alpha-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-gamma production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by alpha-GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer.

We have recently identified alpha-galactosylceramide (alpha-GalCer) as a specific ligand for an invariant Valpha14/Vbeta8.2 T cell receptor exclusively expressed on the majority of Valpha14 NKT cells, a novel subset of lymphocytes. Here, we report that alpha-GalCer selectively activates Valpha14 NKT cells resulting in prevention of tumor metastasis. The effector mechanisms of the ligand-activated Valpha14 NKT cells seem to be mediated by natural killer (NK)-like nonspecific cytotoxicity. Indeed, the cytotoxic index obtained by alpha-GalCer-activated Valpha14 NKT cells was reduced by the addition of cold target tumor cells or by treatment with concanamycin A, which inhibits activation and secretion of perforin, but not by mAbs against molecules involved in the NKT cell recognition and conventional cytotoxicity, such as CD1d, Vbeta8, NK1. 1, Ly49C, Fas, or Fas ligand. These results suggest that the ligand-activated Valpha14 NKT cells kill tumor cells directly through a CD1d/Valpha14 T cell receptor-independent, NK-like mechanism.

A lymphocyte subpopulation, the Vα14 natural killer T (NKT) cells, expresses both NK1.1 and a single invariant T cell receptor encoded by the Vα14 and Jα281 gene segments. Mice with a deletion of the Jα281 gene segment were found to exclusively lack this subpopulation. The Vα14 NKT cell–deficient mice could no longer mediate the interleukin-12 (IL-12)–induced rejection of tumors. Although the antitumor effect of IL-12 was thought to be mediated through natural killer cells and T cells, Vα14 NKT cells were found to be an essential target of IL-12, and they mediated their cytotoxicity by an NK-like effector mechanism after activation with IL-12.

Natural killer T (NKT) lymphocytes express an invariant T cell antigen receptor (TCR) encoded by the Vα14 and Jα281 gene segments. A glycosylceramide-containing α-anomeric sugar with a longer fatty acyl chain (C26) and sphingosine base (C18) was identified as a ligand for this TCR. Glycosylceramide-mediated proliferative responses of Vα14 NKT cells were abrogated by treatment with chloroquine–concanamycin A or by monoclonal antibodies against CD1d/Vβ8, CD40/CD40L, or B7/CTLA-4/CD28, but not by interference with the function of a transporter-associated protein. Thus, this lymphocyte shares distinct recognition systems with either T or NK cells.

NK1.1+ T [natural killer (NK) T] cells express an invariant T cell antigen receptor alpha chain (TCR alpha) encoded by V alpha 14 and J alpha 281 segments in association with a limited number of V betas, predominantly V beta 8.2. Expression of the invariant V alpha 14/J alpha 281, but not V alpha 1, TCR in transgenic mice lacking endogenous TCR alpha expression blocks the development of conventional T alpha beta cells and leads to the preferential development of V alpha 14 NK T cells, suggesting a prerequisite role of invariant V alpha 14 TCR in NK T cell development. In V beta 8.2 but not B beta 3 transgenic mice, two NK T cells with different CD3 epsilon expressions, CD3 epsilon(dim) and CD3 epsilon(high), can be identified. CD3 epsilon(high) NK T cells express surface V alpha 14/V beta 8 TCR, indicating a mature cell type, whereas CD3 epsilon(dim) NK T cells express V beta 8 without V alpha 14 TCR and no significant CD3 epsilon expression (CD3 epsilon(dim)) on the cell surface. However, the latter are positive for recombination activating gene (RAG-1 and RAG-2) mRNA, which are only expressed in the precursor or immature T cell lineage, and also possess CD3 epsilon mRNA in their cytoplasm, suggesting that CD3 epsilon(dim) NK T cells are the precursor of V alpha 14 NK T cells.

A 21-year-old man was referred to our hospital because of a liver mass lesion detected by abdominal ultrasonography. He had received no hormonal treatment. Physical examinations revealed no abnormalities, and laboratory data, including hepatic function test results, were within normal ranges, with the exception of elevated levels of serum protein induced by vitamin K absence or antagonist (PIVKA)-II (2.2 AU/ml). Abdominal ultrasonography revealed a hyperechoic mass lesion measuring 10 x 10 cm, with hypoechoic areas located in the right posterior segment of the liver. A low-density area and a hypervascular area were detected in the right posterior segment of the liver by computed tomography and celiac angiography, respectively. As hepatocellular carcinoma could not be completely excluded, the tumor was resected. The tissue consisted of sheets of tumor cells with eosinophilic cytoplasm and round nuclei showing a thin trabecular pattern, and these histological findings indicated liver cell adenoma. After resection of the tumor, serum PIVKA-II returned to the normal level.