CN101528779A - 4-1 BB ligand in inflammatory diseases - Google Patents

Abstract

The present invention provides 4-1BBL blockers, as well as pharmaceutical compositions and articles of manufacture containing the blockers as novel therapeutic interventions for persistent inflammation. Accordingly, the present invention also provides methods for reducing the persistent production of tumor necrosis factor.

Description

4-1BB ligands in inflammatory diseases

related application

This application claims priority to US Provisional Application 60/852,022, filed October 16, 2006, and US Provisional Application 60/917,561, filed May 11, 2007, both of which are incorporated herein by reference in their entirety.

Statement Regarding Federally Funded Research

Work on this application was supported by US Government grants (GM67101, AI41637 and AI54696). The government has certain rights in this invention.

Background technique

The production of proinflammatory cytokines such as tumor necrosis factor (TNF) in macrophages is important for eliciting innate immune responses and maintaining the systemic inflammatory state that accompanies conditions such as sepsis (Beutler, Nature 430:257-63 (2004) ; Cohen, Nature 420:885-91 (2002)). Toll-like receptors (TLRs) are primary innate immune sensors that each respond to specific molecules of microbial origin (Janeway & Medzhitov, Annu. Rev. Immunol. 20: 197-216 (2002)). TLR4 is a receptor for lipopolysaccharide (LPS) endotoxin by recruiting signaling adapters such as primary response gene for spinal cord differentiation 88 (MyD88) and Toll/interleukin-1 receptor/tolerance adapter protein (TRIF ) (also known as TICAM-1) responds to LPS. This recruitment allows the interaction and activation of IRAK family members and TNF receptor-associated factor 6 (TRAF6), thereby activating the MAP and kinase pathways that control inflammatory cytokine production of NF-κB (Akira & Takeda, Nat. Rev. Immunol .4: 499-511 (2004)).

Although the MAP and kinase pathways of NF-κB are transiently activated in macrophages within hours of lipopolysaccharide (LPS) treatment, the production of pro-inflammatory cytokines such as TNF can persist for up to 24 hours. The production of this inflammatory cytokine is thus part of an inflammatory process characterized by an initiation followed by a "maintenance" phase. This maintenance period is usually terminated after the breakdown of the inflammatory trigger. Sustained cytokine production is important for maintaining the inflammatory state and for the development of inflammatory disease, as many of these events culminate in the disruption of inflammatory regulation that occurs near the end of a sustained inflammatory response. Therefore, sustained TNF production is associated with the pathology of many inflammatory diseases.

Therefore, agents that affect sustained TNF production are useful in the development of therapies for inflammatory diseases.

Contents of the invention

review

The present invention relates to the discovery of the role of 4-1BB ligand (4-1BBL) on the production of pro-inflammatory cytokines that are important in mediating macrophage activation. In particular, the present invention relates to the discovery of the role of 4-1BBL in late signaling events that are important for the sustained production of tumor necrosis factor (TNF) leading to inflammation. The role of 4-1BBL in late cytokine production is dependent on 4-1BB. Accordingly, the present invention relates to novel functions of 4-1BBL dependent on its known receptor 4-1BB and provides methods for reducing inflammatory cytokine production. The invention also relates to 4-1BBL antagonists and blockers, as well as pharmaceutical compositions and articles of manufacture comprising such antagonists and blockers. In addition, the present invention provides methods of screening for 4-1BBL blockers and antagonists.

One aspect of the present invention is a pharmaceutical composition comprising a 4-1BBL blocker and a pharmaceutically acceptable carrier, wherein the 4-1BBL blocker is selected from (a) antagonistic antibodies specific for 4-1BBL; (b ) an oligonucleotide effective to reduce expression of a 4-1BBL nucleic acid in a cell; and (c) soluble 4-1BB. A therapeutically effective amount of the blocking agent may be employed in the composition.

One aspect of the invention is a pharmaceutical combination comprising a 4-1BBL blocker and a second agent that is an anti-inflammatory drug, wherein the 4-1BBL blocker is selected from (a) antagonistic antibodies specific for 4-1BBL; (b) an oligonucleotide effective in reducing expression of a 4-1BBL nucleic acid in a cell; and (c) soluble 4-1BB. A therapeutically effective amount of the blocking agent and/or the anti-inflammatory drug may be employed in the combination. The anti-inflammatory drug may be an antibody specific for tumor necrosis factor.

Another aspect of the invention is a chimeric or humanized antibody specific for 4-1BBL.

Another aspect of the invention is soluble 4-1BB that specifically binds mammalian 4-1BBL. In some embodiments, the mammalian 4-1BBL is human, mouse, rabbit, pig or horse 4-1BBL. In some embodiments, the soluble 4-1BB has amino acids 23 to 186 corresponding to the human 4-1BB polypeptide, amino acids 24 to 187 corresponding to the mouse 4-1BB polypeptide, amino acids corresponding to the human 4-1BB polypeptide 1 to amino acid 186, a sequence corresponding to amino acid 1 to amino acid 187 of the mouse 4-1BB polypeptide or the corresponding region in another mammalian 4-1BB. In some embodiments, the soluble 4-1BB has the sequence of SEQ ID NO: 7, 8, 20 or 21.

Another aspect of the invention is an article of manufacture comprising a container comprising a 4-1BBL blocking agent and instructions for use of the 4-1BBL blocking agent in reducing inflammation and/or reducing inflammatory cytokine production. In some embodiments, the instructions for use of the 4-1BBL blocking agent include instructions for using the 4-1BBL blocking agent in the treatment of inflammatory diseases. A therapeutically effective amount of the blocking agent may be employed in the container.

In some embodiments, the inflammatory disease is rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, psoriasis, ankylosing spondylitis, Crohn's disease, rheumatoid arthritis, systemic juvenile chronic arthritis, osteoarthritis pororiasis, or irritable bowel syndrome.

Another aspect of the invention is a method of reducing inflammatory cytokine production in a mammal comprising administering to the mammal a 4-1BBL blocking agent. The therapeutically marketed blocking agent may be administered. In some embodiments, the method further comprises identifying a mammal having or susceptible to the inflammatory disease. In some embodiments, the mammal is a human, mouse, rabbit, pig or horse.

Another aspect of the invention is a method of reducing inflammation in a mammal comprising administering to the mammal a 4-1BBL blocking agent. The therapeutically marketed blocking agent may be administered. In some embodiments, the mammal is a human, mouse, rabbit, pig or horse.

Another aspect of the present invention is a method of screening for a 4-1BBL blocker comprising (a) contacting cells expressing 4-1BBL with a candidate agent; and (b) detecting whether the candidate agent reduces the production of inflammatory cytokines , or whether the candidate agent prevents 4-1BBL oligomerization, wherein if the candidate agent reduces inflammatory cytokine production or prevents 4-1BBL oligomerization, the candidate agent is a 4-1BBL blocking agent. In some embodiments, the inflammatory cytokine is tumor necrosis factor or IL-6. In some embodiments, the cells are human cells, macrophages and/or RAW264.7 cells.

Another aspect of the present invention is a method of screening for a 4-1BBL blocker comprising (a) administering a candidate agent to a mammal; and (b) detecting whether the candidate agent reduces inflammation or reduces production of inflammatory cytokines, wherein If the candidate agent reduces inflammation or reduces the production of inflammatory cytokines in the mammal, the candidate agent is a 4-1BBL blocker. In some embodiments, the mammal has an inflammatory disease. In some embodiments, the inflammatory cytokine is tumor necrosis factor or is reduced by 20%, 25%, 30% or more than 30%. . In some embodiments, the mammal is a mouse, rat, rabbit or pig.

In some embodiments of the invention, the 4-1BBL is mammalian 4-1BBL. In some embodiments, the 4-1BBL is human, mouse, rabbit, pig or horse 4-1BBL. In some embodiments, the 4-1BBL has the sequence shown in SEQ ID NO: 1 or 2. In this partial embodiment, the blocking agent is a chimeric or humanized antibody specific to 4-1BBL. In some embodiments, the blocking agent is an oligonucleotide having a sequence selected from SEQ ID NO: 10-15, 22-38, and 45-56. In some embodiments, the blocking agent is soluble 4-1BB that binds human 4-1BBL. In some embodiments, the soluble 4-1BB has amino acids 23 to 186 corresponding to the human 4-1BB polypeptide, amino acids 24 to 187 corresponding to the mouse 4-1BB polypeptide, amino acids corresponding to the human 4-1BB polypeptide 1 to amino acid 186 or a sequence corresponding to amino acid 1 to amino acid 187 of the mouse 4-1BB polypeptide. In some embodiments, the blocking agent is soluble 4-1BB having the sequence shown in SEQ ID NO: 7, 8, 20 or 21. In some embodiments, the inflammatory cytokine is tumor necrosis factor or IL-6.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although other methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. As generally understood by those of ordinary skill in the field of the present invention, amino acid codes may include full names, three-letter or single-letter codes. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Description of drawings

Figures 1A-D are results showing that 4-1BBL is a TLR4-interacting protein. (A) To encode amino acids 653-839 of TLR4 and amino acids 5-254 of 4-1BBL (clone 1), amino acids 3-254 of 4-1BBL (clone 2), dominant-negative p38α (AF), p53 or nuclear fiber Growth of yeast cells transfected with expression plasmids for lamin on histidine-deficient media. 4-1BBL, but not an unrelated protein, can interact with the TLR4 intracellular domain. (B)

Immunoassays were performed on 293T cells transfected with empty plasmid (control) or plasmids expressing hemagglutinin-tagged 4-1BBL (HA-4-1BBL) and Flag-tagged TLR4 or IL-3R, after transfection Cell lysis was performed for 24 hours; two-thirds of the lysates were immunoprecipitated (IP) by anti-hemagglutinin (HA). TLR4, but not IL-3R, is pulled down by 4-1BBL. (C) TLR4 expressing GFP-tagged 4-1BBL and Flag-tagged, cytoplasmic domain-deleted TLR4 (TLR4-ΔCyt), IL-3R (left) or Myc-tagged TLR4 or TLR4 with P712H substitution Immunoassay of 293T cells transfected with plasmids of (TLR4-P712H; right); immunoprecipitation of lysates with anti-Flag or anti-Myc. The TLR4 intracellular domain is involved in the interaction with 4-1BBL, and different parts of the TLR4 Toll-IL-1R domain interact with 4-1BBL and MyD88. (D) To express individual Flag-tagged TLR4 and GFP or GFP-tagged 4-1BBL, 4-1BBL with deletion of cytoplasmic domain (4-1BBL-ΔCyt), 4-1BBL with deletion of extracellular domain ( Immunoassay of 293T cells transfected with plasmids of 4-1BBL-ΔExt) or 4-1BBL containing only the cytoplasmic domain (4-1BBL-Cyt). 4-1BBL requires the 4-1BBL cytoplasmic domain and association with the cell membrane to interact with TLR4. IP: immunoprecipitation; IB: immunoblotting. Results are representative of two to three independent experiments.

Figures 2A-D are results showing the in vivo effects of 4-1BBL inhibition. (A) Survival of wild-type and 4-1BBL-deficient mice injected intraperitoneally with 0.5 mg LPS/25 g body weight. 4-1BBL knockout (KO) mice tolerate lethal doses of LPS. Survival of wild-type mice injected intraperitoneally with 300 μg LPS/25 g body weight (B) or 500 μg LPS/25 g body weight (C) in combination with isotype control IgG2a or anti-4-1BBL (250 μg LPS/25 g body weight). The lethal effect of LPS on wild-type mice was reduced by administration of anti-4-1BBL. (D) ELISA for TNF in sera from LPS-injected wild-type or 4-1 BBL-deficient mice. LPS-induced TNF production was greatly reduced in 4-1 BBL KO mice compared to wild-type mice. In (A) and (B) the Log-rank test was used. P=0.0017 in (A); P=0.018 in the left panel of (B); P=0.048 in the right panel of (B). Data in (C) are presented as ± s.d. *, P<0.005, **, P<0.001 (Student's t-test).

Figures 3A-H are results showing the requirement for 4-1BBL for sustained TNF production in LPS-stimulated macrophages. (A) TNF, IL-6 in wild-type (WT) or 4-1BBL-deficient (4-1BBL KO) peritoneal macrophages untreated (none) or treated with LPS (100 ng/mL; LPS) for 24 hours , ELISA for the p40 chain of IL-1β or IL-12 (Il-12p40). Lower right panel, IL-6 production induced by IL-Ιβ (10 ng/mL; control). 4-1BBL-deficient macrophages produced less TNF, IL-6 and IL-12 than wild-type macrophages. (B and C) Culture of wild-type and 4-1BBL-deficient macrophages (B) or 4-1BB-deficient macrophages (4-1BB KO; C) treated with LPS (time, horizontal axis) ELISA produced by TNF in the base. 4-1BBL is critical for late LPS-stimulated TNF production, but 4-1BB is not required to promote sustained LPS-induced TNF production. (D) Quantitative PCR of TNF mRNA in wild-type and 4-1BBL-deficient macrophages treated with LPS (time, horizontal axis). AU: arbitrary units. TNF mRNA in 4-1BBL-deficient and wild-type macrophages peaked at similar amounts two hours after LPS stimulation, but TNF mRNA in 4-1BBL-deficient cells was much lower than that in wild-type cells at the later stage of LPS stimulation . (E) Nuclear transcriptional activity of TNF and glyceraldehyde phosphate dehydrogenase (GAPDH; control) transcription rates in wild-type and 4-1BBL-deficient macrophages stimulated with LPS (100 ng/mL; time, upper bar) RNA dot blot assay (nuclear run-on analysis). LPS-triggered Tnf transcription was present in both 4-1BBL-deficient and wild-type macrophages one hour after LPS stimulation, but Tnf transcription in 4-1BBL-deficient macrophages decreased in the late stage of LPS treatment. (F) Real-time PCR analysis of TNF mRNA stability in wild-type and 4-1BBL-deficient macrophages 3 hours after LPS stimulation. Transcription was inhibited using actinomycin D (10 μg/mL). Values are percentages remaining relative to the value at 0 hours (set to 100%). The deletion of 4-1BBL has a great influence on the stability of TNF mRNA. (G) EMSA of nuclear extracts of LPS (time, upper bar) stimulated wild-type and 4-1BBL-deficient macrophages assessed with radiolabeled probe (left margin). 4-1BBL deletion had no effect on LPS-induced activation of NF-κB or LPS-induced activation of the transcription factor AP-1. (H) Immunoblot of lysates from wild-type or 4-1BBL-deficient macrophages stimulated with LPS (time, upper bar). NF-κB and MAP kinase pathways were not significantly affected by deletion, p-, phosphorylation. ^* , P<0.01, and ^** , P<0.005 vs. control (Student's t-test). Data are mean ± sd (AD) of triplicate and are representative of two to three experiments (AH).

Figures 4A-D are results showing the requirement for 4-1BBL for sustained TNF production in RAW264.7 macrophages. (A) RAW 264.7 cells stably transfected with p-suppressor-containing control siRNA or 4-1BBL-siRNA #1 or #2. Protein levels of 4-1BBL were detected by immunoblotting. (B) TNF production measured in control and 4-1BBL knockdown cells treated with LPS (100 ng/mL) for the indicated times. (C) The control and 4- 1BBL inhibits TNF production in cells. Both siRNAs potently inhibited 4-1BBL expression in RAW264.7 cells (A) and inhibited LPS- and other TLR ligand-induced TNF production (B and C). 4-1BBL has no effect on LPS-induced NF-κB activation in RAW264.7 cells as reflected by I-κB-α degradation (D). Data are shown as mean ± s.d. of three independent experiments in duplicate. *, P<0.05, and **, P<0.01 vs. control. Results in (a) are representative of 3 experiments.

Figures 5A-B are results showing that 4-1BB is not involved in LPS-induced TNF production in RAW264.7 macrophages. (A) RAW 264.7 cells stably transfected with p-suppressor-containing control siRNA, 4-1BBL-siRNA #1 or #2. 4-1BB was detected by RT-PCR. (B) TNF production measured in control and 4-1BBL cells treated with LPS (100 ng/mL) for 24 hours. Small interfering RNA effectively inhibited 4-1BB production in RAW264.7 cells (A), but no effect was observed on LPS-induced TNF production (B). Data are shown as mean ± s.d. of three independent experiments in duplicate.

Figure 6A-D is a result showing that 4-1BB is involved in TLR-ligand-induced TNF generation in macrophages (A) combined with empty (control) or HA-4-1BBL expression vector Flag-TLR2, Flag-TLR3, Flag-TLR4, or Flag-TLR9 transfected 293T cells. Cells were lysed 24 hours after transfection. Cell lysates were immunoblotted with anti-Flag antibody, immunoprecipitated with anti-HA followed by anti-HA and anti-flag antibodies. All tested TLRs were pulled down by 4-1BBL in the co-immunoprecipitation assay. (B) Cultured with Pam3 (1 μg/mL), poly I:C (25 μg/mL), LPS (100 ng/mL), R848 (100 nM), CpG (5 μg/mL), IL-1β (10 ng/mL) or Basal (none) treatment of wild-type and 4-1BBL-deficient macrophages. TNF production was measured 24 hours after treatment. Pam3(TLR2 ligand)-, PolyI:C(TLR3 ligand)-, R848(TLR7&8 ligand)-, CpG(TLR9 ligand)-induced TNF production was reduced in 4-1BBLKO cells. (C) Wild-type and TLR4-deficient macrophages were treated with 0, 1, 2.5 or 5 μg/mL and incubated for 24 hours, or (D) were treated with 1 μg/mL of CpG DNA and supernatants were collected at indicated time points solution to measure TNF production. There was a small but statistically significant decrease in TLR9-induced TNF production in TLR4-deficient macrophages with low doses of CpG (1 μg/mL). Data are presented as mean ± s.d. of triplicate. *, P<0.05, **, P<0.01, ***, P<0.005. Results represent two to four experiments.

Figures 7A-F are results showing the requirement for 4-1BBL induction and cell surface localization for sustained TNF production in LPS-treated macrophages. (A) Immunity of 4-1BBL in lysates from wild-type macrophages treated with LPS (time, upper bars) and macrophages deficient in TLR4 (TLR4KO), MyD88 (MyD88KO), or TRIF (TRIF KO) blot. LPS induced rapid expression of 4-1BBL in macrophages in a TLR4-, MyD88- and TRIF-dependent manner. (B) Wild-type macrophages treated with no pretreatment (None) or with inhibitors of NF-κB (20 μM sulfasalazine), p38 (10 μM SB203580), Jnk (5 μM SP600125) or MEK (10 μM PD98059) for 1 h. Phage cells, semi-quantitative PCR of 4-1BBL mRNA after treatment with LPS (time, upper bar). LPS-induced 4-1BBL mRNA was efficiently inhibited by the NF-κΒ inhibitor sulfasalazine and the proteasome inhibitor MG-132 (data not shown). In addition, the p38 inhibitor SB203580, the Jnk inhibitor SP600125 and the Mek inhibitor PD98059 also had a certain inhibitory effect on the expression of 4-1BBL. (C) Stability of LPS-induced 4-1BBL mRNA (treatment for 1 hour; LPS) or ectopically expressed 4-1BBL mRNA after infection with recombinant adenovirus (Adv) encoding 4-1BBL for 18 hours real-time PCR analysis. Actinomycin D can be used to inhibit transcription. Values are percentages remaining relative to the value at 0 hours (set to 100%). LPS-induced changes in mRNA stability may also be involved in LPS-induced 4-1BBL expression. (D) Flow cytometry of 4-1BBL expression in wild-type peritoneal macrophages treated with LPS (time, legend): right, surface expression; left, in cells permeabilized with 0.1% saponin total expression. LPS-induced 4-1BBL localizes on the cell surface. (E) Wild-type peritoneal macrophages were pre-incubated with (+) or without (-) brefeldin A (3 μg/mL) for 1 h, then treated with LPS (time, left margin) and treated with anti- Immunofluorescence microscopy after 4-1BBL immunostaining. Initial magnification x 100. Brefeldin A blocks protein cell surface translocation by inhibiting protein translocation from the endoplasmic reticulum to the Golgi apparatus. (F) Semi-quantitative PCR of 4-1BBL and TNF mRNA in lysates of cells treated as described in E. Brefeldin A affected neither the LPS-induced increase in 4-1BBL mRNA nor the LPS-induced TNF mRNA after 1 hour, but decreased TNF mRNA at 2, 4 and 6 hours. GAPDH (A, B, F): glyceraldehyde phosphate dehydrogenase (loading control). Data represent three independent experiments.

Figures 8A-F are results showing that TLR ligands, but not IL-Ιβ, induce 4-1BBL expression in macrophages. Wild-type macrophages were treated with LPS, IL-1β, poly I:C, peptidoglycan, CpG DNA, or R848 for indicated periods of time. Culture supernatants of untreated cells treated with LPS or IL-1β for 24 hours were harvested, and IL-6 levels were determined by ELISA. Data in (B) are presented as mean ± s.d. of triplicate. 4-1BBL expression can be analyzed by Western blot using anti-4-1BBL antibody. GAPDH was used as a control. Results are representative of two to three experiments.

Figures 9A-G are results showing that expression and crosslinking of 4-1BBL trigger TNF production. Wild-type macrophages (A), wild-type and 4-1BB-deficient macrophages (B ), wild-type and TLR-deficient macrophages (C), wild-type and TLR-2-deficient macrophages (D) or wild-type and TRF-deficient macrophages (E) produced 4-1BBL Western blot analysis (upper panel) and ELISA analysis of TNF (lower panel). PFU: plaque forming unit. 4-1BBL expression induces TNF production in a dose-dependent manner (A) that does not require 4-1BB (B). TNF induction was partially impaired in macrophages isolated from TLR4-/- mice (C) and decreased in TLR2-deficient macrophages (D). TNF induction was independent of TRIF, as TRIF deficiency had no effect on 4-1BBL-induced TNF production (E). (F) Wild-type macrophages treated with goat anti-human Fc (anti-Fc; 1.5 μg/mL), 4-1BB-Fc (5 μg/mL) or medium alone (none) for 24 hours ELISA of TNF produced by cells or macrophages deficient in 4-1BBL, MyD88, TRIF, TLR2, TLR4, or both MyD88 and TRIF. Cross-linking of more than two 4-1BBL molecules is required to initiate TNF production. (G) With medium alone (none) or LPS or with Fc, 4-1BB-Fc or anti-4-1BBL (α-4-1BBL; 0, 2 or 5 μg/mL, respectively, isotype antibody IgG2a added to total ELISA of TNF production in wild-type macrophages incubated at 5 μg/mL for 24 hours. Both 4-1BB-Fc and anti-4-1BBL antibodies inhibited LPS-induced TNF production. ^* , P<0.05, ^** , P<0.01, ^*** , P<0.005, relative to control (Student's t-test). Data are mean±s.d. of triplicate samples and are representative of two to four experiments.

Figures 10A-I show the presence of two consecutive TLR4 complexes. (A) Immunoassay of wild-type macrophages treated with LPS (time, upper bar); total cell lysate (lysate; upper) or immunoprecipitated with anti-TLR4, anti-MyD88, or anti-4-1BBL The lysates can be analyzed by immunoblotting with anti-4-1BBL, anti-TLR4 and/or anti-MyD88. Dashed box, isotype antibody (negative control for immunoprecipitation); solid box, positive control for immunoblotting of 4-1BBL or MyD88. In LPS-treated macrophages, the MyD88-TLR4 complex is responsible for the initial cellular response, whereas the 4-1BBL-TLR4 complex is involved in sustained TNF production. (B) Flag-TLR4, but not flag-4-1BBL, is pulled down by MyD88, showing that MyD88 cannot interact with 4-1BBL. (C) 4-1BBL interacts with TRAF6 but not TRAF2. (D) Inhibition of TRAF6 impaired LPS, poly I:C and IL-1β-induced cytokine production, but had no effect on TNF-induced IL-6 production. (E) EMSA of nuclear extracts of wild-type macrophages infected with adenoviruses expressing GFP (Adv-GFP) or 4-1BBL (Adv-4-1BBL) (time, upper bar), radiolabeled Probe analysis of (left margin). LPS (bottom right), LPS-treated samples (positive control for NF-κB). Expression of 4-1BBL activates CREB and C/EBP, but not NF-κB. (F) Immunoblot of various proteins (left margin) in total lysates of cells described in E. (G) Infected with adenovirus expressing 4-1BBL and treated with inhibitors of NF-κB (20 μM sulfasalazine), p38 (10 μM SB203580), Jnk (5 μM SP600125) or MEK (10 μM PD98059) 6 hours after infection , and wild-type macrophages were analyzed by ELISA for TNF production at 24 hours post-infection. ^* , P<0.01, ^** , P<0.005, versus control (Student's t-test). (H) Top, semi-quantitative PCR of TNF mRNA from wild-type macrophages infected (time, upper bar) with adenovirus expressing 4-1BBL. Bottom, TNF mRNA stability in wild-type macrophages infected with adenovirus for 18 hours or treated with LPS for 1 hour. Actinomycin D can be used to inhibit transcription. Values are percentages remaining relative to the value at 0 hours (set to 100%). Data represent mean ± sd (G) of three experiments and represent two (A, G, H), three (E) or four (F) experiments. No degradation of IκBα was observed in cells overexpressing 4-1BBL (F). Expression of 4-1BBL triggers partial phosphorylation of p38, Jnk and Erk (F) and inhibits 4-1BBL-mediated TNF production with reduction of p38, Jnk and Erk (but not inhibition of NF-κB) (G) . Deletion of 4-1BBL resulted in less LPS-induced TNF mRNA transcription at a later stage, whereas 4-1BBL overexpression resulted in more TNF mRNA (H). TNF transcripts have similar half-lives (H) in 4-1BBL-overexpressing and LPS-treated cells. (I) Model of continuous signal transduction in LPS-treated macrophages. TLR4-LPS binding leads to MyD88- and TRIF-dependent expression of inflammatory genes including TNF within hours. 4-1BBL is induced by this early response and translocates to the cell surface to interact with TLRs and initiate signal transduction for the sustained expression of inflammatory genes such as TNF.

Figures 11A-F summarize data showing the relationship between 4-1BBL and IL-6 induction. 4-1BBL-/- mice produced significantly less IL-6 than wild-type mice in response to LPS (A). Macrophages from wild-type mice produced significantly more IL-6 than macrophages from 4-1BBL-/- mice (B). Wild-type macrophages treated with Pam3, poly I:C, LPS, R848 and CpG produced significantly more IL-6 than 4-1BBL-/- macrophages treated with the same inducers (C). Macrophages incubated with LPS and 4-1BB-Fc produced significantly less IL-6 than macrophages incubated with LPS alone, whereas macrophages incubated with LPS and increasing concentrations of anti-4-1BBL antibody Decreased IL-6 production levels are shown (D). RAW264.7 cells whose expression of 4-1BBL was significantly reduced by 4-1BBL-specific siRNA produced significantly less IL-6 than RAW264.7 cells treated with control siRNA (E). 4-1BBL expression was significantly reduced by 4-1BBL-specific siRNA and expressed as peptidoglycan (PG, 10 μg/mL), poly I:C (25 μg/mL), LPS (100 ng/mL), CpG (5 μg/mL) ) treated cells produced more Significantly less IL-6 was produced (Fig. 1 IF).

Figure 12 is a schematic diagram showing the domains of mouse 4-1BBL (A) and human 4-1BBL (B). "Cyt" refers to the cytoplasmic domain, "TM" refers to the transmembrane domain, "Ext" refers to the extracellular domain, and "Sig" refers to the signal peptide.

Figure 13 is a schematic diagram showing the mouse 4-1BB domain (A), human 4-1BB domain (B) and soluble mouse 4-1BB-human Ig-Fc fusion protein (C). "Cyt" refers to the cytoplasmic domain, "TM" refers to the transmembrane domain, "Ext" refers to the extracellular domain, and "Sig" refers to the signal peptide.

Detailed description of the invention

The present invention relates to the discovery of the role of 4-1BB ligand (4-1BBL) on the production of pro-inflammatory cytokines in macrophage activation. In particular, the present invention relates to the discovery of the role of 4-1BBL in late signaling events that can lead to the sustained production of inflammatory cytokines such as TNF leading to excessively persistent inflammation. The role of 4-1BBL in late signal transduction is dependent on 4-1BB. Therefore, the present invention provides a 4-1BBL blocking agent, and pharmaceutical compositions and pharmaceutical compositions containing the blocking agent capable of reducing the production of inflammatory cytokines (such as TNF and IL-6) in a manner independent of 4-1BB. Manufacture items. The invention provides a method for reducing inflammation, a method for reducing the production of inflammatory cytokines, and a method for screening 4-1BBL blockers.

4-1BBL polypeptide and nucleic acid

The studies described here show that the initial induction of TNF expression and sustained TNF production in macrophages is regulated by early and late signal transduction timing. Toll-like receptor (TLR) 4-mediated TNF production in macrophages usually occurs within hours and is maintained for almost a day (see Galanos et al. Proc.Natl.Acad.ScL USA 76:5939-43 (1979 )). Sustained TNF production involves TLR-signaling controlled by the cell surface 4-1BB ligand (4-1BBL). This 4-1BBL function is independent of myeloid differentiation primary response gene 88 (MyD88) and Toll/interleukin-1 receptor/tolerance adapter protein (TRIF), but is dependent on TNF receptor-associated factor 6 (TRAF6). Therefore, the signalsome TLR4/MyD88/TRIF is responsible for the initiation and early expression of inflammatory genes, while 4-1BBL is responsible for the sustained TNF production in the later stage. 4-1BBL is one of the early induced proteins in macrophages in response to inflammatory stimuli and is induced only in the early stages of macrophage activation. Newly synthesized 4-1BBL translocates to the cell surface to form a new signaling phase for later sustained TNF production. Therefore, diseases associated with the overproduction of inflammatory cytokines (for example, TNF or IL-6) or with the regulation failure of sustained inflammatory response can be obtained by inhibiting or reducing the activity of 4-1BBL polypeptide or the expression of 4-1BBL nucleic acid. Medicines for treatment.

4-1BBL (4-1BB ligand) is a member of the TNF family of type II cell surface glycoproteins expressed on activated antigen-presenting cells such as activated B cells and macrophages. 4-1BBL polypeptides are known in the art and may be derived from any source, for example, from mouse, rabbit, pig, dog, cow, monkey or human. An example of mouse 4-1BBL polypeptide is as follows (GenBank accession number NP_033430):

1 MDQHTLDVED TADARHPAGT SCPSDAALLR DTGLLADAAL LSDTVRPTNA

51 ALPTDAAYPA VNVRDREAAW PPALNFCSRH PKLYGLVALV LLLLIAACVP

101 IFTRTEPPRPA LTITTSPNLG TRENNADQVT PVSHIGCPNT TQQGSPVFAK

151 LLAKNQASLC NTTLNWHSQD GAGSSYLSQG LRYEEDKKEL VVDSPGLYYV

201 FLELKLSPTF TNTGHKVQGW VSLVLQAKPQ VDDFDNLALT VELFPCSMEN

251 KLVDRSWSQL LLLKAGHRLS VGLRAYLHGA QDAYRDWELS YPNTTSFGLF

301 LVKPDNPWE (SEQ ID NO: 1)

An example of the human 4-1BBL polypeptide is as follows (GenBank accession number P41273):

1 MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA

51 CPWAVSGARA SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV

101 LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR

151 RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ

201 GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS

251 PRSE (SEQ ID NO: 2)

4-1BBL can be identified based on function or sequence. For example, although the present invention is based in part on the discovery that 4-1BBL mediates TNF production in a 4-1BB-independent manner in macrophages, 4-1BBL is also known to interact with 4-1BB (in activated CD4 and Members of the TNF receptor superfamily of transmembrane proteins expressed on CD8 T cells) interact to generate co-stimulatory signals during T cell receptor activation. Watts, Annu. Rev. Immunol. 23:23-68 (2005). Furthermore, Figure 12 shows the functional domains of human and mouse 4-1BBL.

As used herein, "4-1BBL" or "4-1BBL polypeptide" includes any biologically active fragment of the full-length polypeptide, as well as any fragment suitable for use as an immunogen to raise antibodies against biologically active 4-1BBL. Accordingly, a 4-1BBL polypeptide may have an amino acid sequence substantially identical to that shown in SEQ ID NO: 1 or 2. In general, the term "substantially identical" means that an amino acid sequence differs from a reference sequence only by conservative amino acid substitutions, e.g., the substitution of one amino acid for another of the same type (e.g., valine for glycine, arginine for lysine ), or differ by one or more non-conservative substitutions, deletions or insertions at amino acid sequence positions that do not disrupt the function of the protein. If the polypeptide (or nucleic acid) sequence exhibits about 50% homology to the reference sequence, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 95% % homology, it is basically consistent with the reference sequence. For example, the 4-1BBL polypeptide can be at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than 90% identical to SEQ ID NO: 1 and 2 sex.

A 4-1BBL nucleic acid is a polymer of DNA or RNA encoding a 4-1BBL polypeptide. 4-1BBL nucleic acid can be genomic DNA and messenger RNA. It can be incorporated into plasmid vectors or viral DNA. It can be single or double stranded, circular or linear. Examples of 4-1BBL nucleic acids include, but are not limited to, nucleic acids encoding the mouse and human 4-1BBL polypeptide sequences shown in SEQ ID NO.1 and 2. An exemplary 4-1BBL nucleic acid encoding a mouse 4-1BBL polypeptide (SEQ ID NO: 1) is the following sequence, which is available from GenBank under accession number NM_009404:

1 atggaccagc acacacttga tgtggaggat accgcggatg ccagacatcc

51 agcaggtact tcgtgcccct cggatgcggc gctcctcaga gataccgggc

101 tcctcgcgga cgctgcgctc ctctcagata ctgtgcgccc cacaaatgcc

151 gcgctcccca cggatgctgc ctaccctgcg gttaatgttc gggatcgcga

201 ggccgcgtgg ccgcctgcac tgaacttctg ttcccgccac ccaaagctct

251 atggcctagt cgctttggtt ttgctgcttc tgatcgccgc ctgtgttcct

301 atcttcaccc gcaccgagcc tcggccagcg ctcacaatca ccacctcgcc

351 caacctgggt acccgagaga ataatgcaga ccaggtcacc cctgtttccc

401 acattggctg ccccaacact acacaacagg gctctcctgt gttcgccaag

451 ctactggcta aaaaccaagc atcgttgtgc aatacaactc tgaactggca

501 cagccaagat ggagctggga gctcatacct atctcaaggt ctgaggtacg

551 aagaagacaa aaaggagttg gtggtagaca gtcccgggct ctactacgta

601 tttttggaac tgaagctcag tccaacattc acaaacacag gccacaaggt

651 gcagggctgg gtctctcttg ttttgcaagc aaagcctcag gtagatgact

701 ttgacaactt ggccctgaca gtggaactgt tcccttgctc catggagaac

751 aagttagtgg accgttcctg gagtcaactg ttgctcctga aggctggcca

801 ccgcctcagt gtgggtctga gggcttatct gcatggagcc caggatgcat

851 acagagactg ggagctgtct tatcccaaca ccaccagctt tggactcttt

901 cttgtgaaac ccgacaaccc atgggaatga (SEQ ID NO: 3)

An exemplary 4-1BBL nucleic acid encoding a human 4-1BBL polypeptide (SEQ ID NO: 2) is the following sequence, which is available from GenBank under accession number U03398:

1 gtcatggaat acgcctctga cgcttcactg gaccccgaag ccccgtggcc

51 tcccgcgccc cgcgctcgcg cctgccgcgt actgccttgg gccctggtcg

101 cggggctgct gctgctgctg ctgctcgctg ccgcctgcgc cgtcttcctc

151 gcctgcccct gggccgtgtc cggggctcgc gcctcgcccg gctccgcggc

201 cagcccgaga ctccgcgagg gtcccgagct ttcgcccgac gatcccgccg

251 gcctcttgga cctgcggcag ggcatgtttg cgcagctggt ggcccaaaat

301 gttctgctga tcgatgggcc cctgagctgg tacagtgacc caggcctggc

351 aggcgtgtcc ctgacggggg gcctgagcta caaagaggac acgaaggagc

401 tggtggtggc caaggctgga gtctactatg tcttctttca actagagctg

451 cggcgcgtgg tggccggcga gggctcaggc tccgtttcac ttgcgctgca

501 cctgcagcca ctgcgctctg ctgctggggc cgccgccctg gctttgaccg

551 tggacctgcc acccgcctcc tccgaggctc ggaactcggc cttcggtttc

601 cagggccgct tgctgcacct gagtgccggc cagcgcctgg gcgtccatct

651 tcacactgag gccagggcac gccatgcctg gcagcttacc cagggcgcca

701 cagtcttggg actcttccgg gtgacccccg aaatcccagc cggactccct

751 tcaccgaggt cggaataacg cccagcctgg gtgcagccca cctggacaga

801 gtccgaatcc tactccatcc ttcatggaga cccctggtgc tgggtccctg

851 ctgctttctc tacctcaagg ggcttggcag gggtccctgc tgctgacctc

901 cccttgagga ccctcctcac ccactccttc cccaagttgg accttgatat

951 ttattctgag cctgagctca gataatatat tatatatatt atatatatat

1001 atatatttct atttaaagag gatcctgagt ttgtgaatgg acttttttag

1051 aggag ttgt ttgggggggg ggtcttcgac attgccgagg ctggtcttga

1101 actcctggac ttagacgatc ctcctgcctc agcctcccaa gcaactggga

1151 ttcatccttt ctattaattc attgtactta tttgcctatt tgtgtgtatt

1201 gagcatctgt aatgtgccag cattgtgccc aggctagggg gctatagaaa

1251 catctagaaa tagactgaaa gaaaatctga gttatggtaa tacgtgagga

1301 atttaaagac tcatccccag cctccacctc ctgtgtgata cttgggggct

1351 agcttttttc tttctttctt ttttttgaga tggtcttgtt ctgtcaacca

1401 ggctagaatg cagcggtgca atcatgagtc aatgcagcct ccagcctcga

1451 cctcccgagg ctcaggtgat cctcccatct cagcctctcg agtagctggg

1501 accacagttg tgtgccacca cacttggcta actttttaat ttttttgcgg

1551 agacggtatt gctatgttgc caaggttgtt tacatgccag tacaatttat

1601 aataaacact catttttcc (SEQ ID NO: 4)

The 4-1BBL nucleic acid can also encode fragments of the polypeptide sequences shown in SEQ ID NO.1 and 2, provided that the nucleic acid encodes a biologically active polypeptide or fragment suitable for use as an immunogen to form antibodies against biologically active 4-1BBL as discussed above .

General 4-1BBL blocker

A 4-1BBL blocker can be a large or small molecule that inhibits or reduces the expression and/or activity of 4-1BBL. 4-1BBL blockers can inhibit or reduce the activity of 4-1BBL. Examples of such 4-1BBL blocking agents include, but are not limited to, 4-1BBL-specific antibodies, soluble forms of 4-1BB, and agents or small molecules that interfere with protein translocation to the cell surface, such as Brefeldia Prime A. These 4-1BBL blockers may interfere with 4-1BBL oligomerization and/or interfere with the interaction of 4-1BBL with appropriate signaling molecules (eg, TLR and TRAF6) in later signaling events involved in TNF production. Action forms action. The term "4-1BBL oligomerization" as used herein refers to the formation of aggregates of 4-1BBL molecules required for 4-1BBL-mediated production of inflammatory cytokines (eg, TNF or IL-6). 4-1 oligomerization refers to the formation of aggregates of more than two 4-1BBL molecules, eg, aggregates of three to four 4-1BBL molecules. 4-1BBL blockers interfere with the translocation of the protein to the cell surface, as cell surface localization of 4-1BBL is required for proper functioning during later stages of signal transduction.

The 4-1BBL blocker can also act by inhibiting or reducing the expression of 4-1BBL nucleic acid. 4-1BBL blockers can act at the transcriptional or translational level, thereby altering the amount of 4-1BBL produced by cells. 4-1BB blockers can reduce the production of 4-1BBL mRNA transcripts or reduce the stability of the mRNA transcripts. These blocking agents include, but are not limited to, oligonucleotides such as antisense RNA, siRNA and ribozymes. The oligonucleotide-based 4-1BBL blocking agent can be used with 4-1BBL under physiological conditions (for example, about 37 ° C, pH 7 to 7.8, and electrolytes at physiological concentrations) or under stringent or highly stringent conditions. The nucleic acid hybridizes, and inhibits or reduces the expression of the 4-1BBL nucleic acid. The blocking agent may also include inhibitors of other molecules involved in 4-1BBL expression or activity.

Oligonucleotides are polymers of ribonucleotides or deoxyribonucleotides greater than 3 nucleotides in length. Oligonucleotides may comprise naturally occurring nucleotides; synthetic, modified, or pseudonucleotides, such as phosphorothioate; and nucleotides with detectable labels, such as ^P32 , biotin, or digoxigenin. Nucleotides.

An oligonucleotide capable of reducing expression of a 4-1BBL nucleic acid, ie, an oligonucleotide of the present invention, may be fully complementary to the 4-1BBL nucleic acid. Alternatively, some variation may be tolerated between sequences. An oligonucleotide capable of hybridizing to a 4-1BBL nucleic acid under physiological conditions (e.g., physiological temperature and salt concentration) or under stringent or highly stringent hybridization conditions has sufficient complementarity to inhibit expression of the 4-1BBL nucleic acid . Generally, stringent hybridization conditions can be selected to be about 5°C lower than the thermal melting point ( _Tm ) at a specific ionic strength pH. However, stringent conditions include temperatures in the range of about 1°C to about 20°C below the thermal melting point of the selected sequence, depending on the degree of target stringency otherwise specified herein. Contains, for example, 2, 3, 4 or 5 or more contiguous nucleotide stretches that are exactly complementary to the 4-1BBL coding sequence, and are each separated by contiguous nucleotide stretches that are not complementary to the contiguous coding sequence An inhibitory oligonucleotide that inhibits the function of 4-1BBL nucleic acid. Generally, each stretch of adjacent nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary inserts may be 1, 2, 3 or 4 nucleotides in length. Those skilled in the art can easily estimate the degree of mismatch that can be tolerated in inhibiting the expression of a specific target nucleic acid by calculating the melting point of the oligonucleotide hybridized to the sense nucleic acid. Oligonucleotide-based 4-1BBL blockers of the present invention include, for example, small interfering RNA (siRNA), antisense nucleic acid or ribozyme.

The amount of the 4-1BBL blocker to inhibit or reduce the expression and/or activity of 4-1BBL can be, for example, 2%, 5%, 10%, 20%, 40%, 60%, 80%, 95% or 100% %. The activity of 4-1BBL can be determined by methods known in the art, including methods described herein, such as, but not limited to, measuring sustained TNF production, detecting whether 4-1BBL interacts with TLR or TRAF6, for example, measuring 4-1BBL - Whether 1BBL is on the cell surface. Expression of 4-1BBL can also be determined by methods known in the art, including, but not limited to, northern blots to detect levels of 4-1BBL transcripts or western blots to detect levels of 4-1BBL polypeptides.

Examples of the various types of 4-1BBL blockers are discussed in detail below.

4-1BBL-specific antibody

The 4-1BBL blocker can be an antagonistic antibody directed against a 4-1BBL polypeptide. The term "antibody" refers to immunoglobulin molecules, such as monoclonal antibodies, as well as immunologically active portions of immunoglobulin molecules. Monoclonal antibodies are populations of antibody molecules that specifically bind to a specific antigenic epitope. Immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments. Anti-4-1BBL antibodies can be, but are not limited to, murine, chimeric, humanized and/or fully human antibodies. Murine antibodies are antibodies derived entirely from murine sources, eg, antibodies from murine hybridomas formed by the fusion of mouse myeloma cells and mouse B-lymphocytes. Chimeric antibodies are antibodies in which the variable regions are derived from a non-human source (eg, murine or primate) and the constant regions are derived from a human source. A humanized antibody has the antigen-binding regions (eg, determinant complementarity regions) derived from a mouse, while the remaining variable and constant regions are derived from an antibody of human origin. Fully human antibodies are antibodies derived from human cells or from transgenic mice carrying human antibody genes.

An antibody directed against a 4-1BBL polypeptide is a 4-1BBL-specific antibody, thus, it will bind a 4-1BBL polypeptide and not bind a non-4-1BBL polypeptide. 4-1BBL-specific antibodies are antagonistic antibodies that interfere with or block proper 4-1BBL function. See, eg, Figures 2A-D. Antagonistic antibodies can, for example, interfere with the oligomerization of two or more 4-1BBLs, or block the binding of downstream signaling molecules (e.g., TLR or TRAF6) involved in sustained TNF production, thereby reducing the production of inflammatory cytokines. generate. For example, such antagonistic antibodies can bind two 4-1BBLs and prevent the formation of aggregates of more than two 4-1BBLs required to mediate cytokine production. This antagonistic antibody also binds to a specific region on 4-1BBL, thereby preventing its interaction with downstream signaling molecules required to mediate cytokine production (e.g., TLR2, TLR3, TLR4, TLR7, TLR8, TLR9, and TRAF6) combination.

Whether an anti-4-1BBL antibody is an antagonist antibody can be determined by the methods disclosed herein. For example, the production of inflammatory cytokines (eg, TNF) can be assayed in the presence or absence of the antibody. An antibody is an antagonistic antibody if its presence results in a decrease in the level or duration of inflammatory cytokine production.

Methods of generating antibodies are well known in the art. For example, 4-1BBL polyclonal antibodies can be prepared by immunizing a suitable mammal with isolated 4-1BBL polypeptide or an antigenic fragment of the 4-1BBL polypeptide. The mammal can be, for example, a rabbit, goat or mouse. The 4-1BBL polypeptide or antigen fragment can be expressed by recombinant DNA technology, prepared by chemical synthesis, or purified by standard protein purification technology. At an appropriate time after immunization, antibody molecules can be isolated from the mammal (e.g., from blood or other bodily fluids of the mammal) and further analyzed using standard techniques (including, but not limited to, ammonium sulfate precipitation, gel filtration chromatography , ion exchange chromatography or affinity chromatography using protein A) for purification. In addition, antibody-producing cells of such mammals can be isolated and used to prepare hybridoma cells secreting 4-1BBL monoclonal antibodies. Techniques for preparing monoclonal antibody-secreting hybridoma cells are known in the art. See, eg, Kohler and Milstein, Nature 256:495-97 (1975) and Kozbor et al. Immunol Today 4:72 (1983). 4-1BBL monoclonal antibodies can also be prepared by other methods known in the art, eg, expression from recombinant DNA molecules, or screening of recombinant combinatorial immunoglobulin libraries by 4-1BBL polypeptides.

Methods for producing chimeric and humanized monoclonal antibodies are also well known in the art and include, for example, methods involving recombinant DNA techniques. Chimeric antibodies can be produced by expression of nucleic acids encoding non-human variable regions and human constant regions of the antibody molecule. See, eg, Morrison et al., Proc. Nat. Acad. Sci. U.S.A. 86:6851 (1984). Humanized antibodies can be produced by expression of nucleic acids encoding non-human antigen binding regions (determinant complementary regions) and human variable regions (no antigen binding regions) and human constant regions. See, eg, Jones et al., Nature 321:522-24 (1986); and Verhoeven et al., Science 239:1534-36 (1988). Fully human antibodies can be produced by immunization of transgenic mice engineered to express only human heavy and light chain genes. In this case, conventional hybridoma technology or monoclonal antibodies of therapeutic use can be obtained. See, eg, Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). Nucleic acids and techniques involved in antibody design and production are well known in the art. See, e.g., Batra et al., Hybridoma 13:87-97 (1994); Berdoz et al., PCR Methods Appl. 4:256-64 (1995); Boulianne et al. Nature 312:643-46 (1984); Carson et al. People, Adv.Immunol.38:274-311 (1986); People such as Chiang, Biotechniques 7:360-66 (1989); People such as Cole, MoI.Cell.Biochem.62:109-20 (1984); Jones et al. People, Nature 321: 522-25 (1986); Larrick et al., Biochem Biophys. Res. Commun. 160: 1250-56 (1989); Morrison, Annu. Rev. Immunol. 10: 239-65 (1992); Morrison et al. People, Proc.Nat'l Acad.ScL USA 81:6851-55 (1984); Orlandi et al., Pro.Nat'l Acad.Sci.U.S.A.86:3833-37 (1989); Sandhu, Crit.Rev.Biotechnol 12: 437-62 (1992); Gavilondo and Larrick, Biotechniques 29: 128-32 (2000); Huston and George, Hum. Antibodies. 10: 127-42 (2001); Kipriyanov and Le Gall, MoI. Biotechnol. 26:39-60 (2004).

soluble 4-1BB

The 4-1BBL blocker can also be a soluble 4-1BB molecule. 4-1BB is a member of the TNF receptor family of type I transmembrane proteins expressed on activated T cells. See Watts, Annu. Rev. Immunol. 23:23-68 (2005). An exemplary mouse 4-1BB is the following sequence, which is available at GenBank under accession number NP 035742:

1 MGNNCYNVVV IVLLLVGCEK VGAVQNSCDN CQPGTFCRKY NPVCKSCPPS

51 TFSSIGGQPN CNICRVCAGY FRFKKFCSST HNAECECIEG FHCLGPQCTR

101 CEKDCRPGQE LTKQGCKTCS LGTFNDQNGT GVCRPWTNCS LDGRSVLKTG

151 TTEKDVVCGP PVVSFSPSTT ISVTPEGGPG GHSLQVLTLF LALTSALLLA

201 LIFITLLFSV LKWIRKKFPH IFKQPFKKTT GAAQEEDACS CRCPQEEEGG

251 GGGYEL (SEQ ID NO: 5)

An exemplary human 4-1BB is the following sequence, which is available at GenBank under accession number NP_001552:

1 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP

51 NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHLCGAGCS

101 MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG

151 TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL

201 FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE

251 GGCEL (SEQ ID NO: 6)

As shown in FIG. 13 for mouse and human polypeptides, a 4-1BB polypeptide generally includes a signal sequence, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

Soluble 4-1BB refers to a 4-1BB polypeptide that, as a transmembrane protein, does not bind to the cells that produce it, and is capable of inhibiting and/or reducing the activity of 4-1BBL. For example, soluble 4-1BB can be a single polypeptide consisting of the extracellular domain of full-length 4-1BB. As used herein, "full-length 4-1BB" is a polypeptide expressed by a cell having an endogenous nucleic acid encoding it. Full-length 4-1BB has an extracellular domain, a transmembrane domain and a cytoplasmic domain. It may or may not have a signal peptide. Conversely, soluble 4-1BB can be any form of 4-1BB that is not full-length, at least it does not bind cells as a transmembrane protein. Exemplary of a soluble 4-1BB polypeptide is a polypeptide corresponding to the extracellular domain of a full-length polypeptide, or a polypeptide composed of a C-terminus and an unrelated polypeptide fragment (such as the Fc fragment of an immunoglobulin molecule, e.g., an IgG ₁ Fc fragment, or any Conventional protein tags or fusion polypeptides consisting of fusion moieties such as GFP, HA, and 4-1BB ectodomain fused to FLAG and GST). Soluble 4-1BB can be multiple polypeptides or dimers, such as the mouse 4-1BB/Fc discussed in the Examples. An example of a soluble 4-1BB that is also a dimer is the mouse 4-1BB/human Ig-Fc polypeptide discussed in the Examples section.

An example of a soluble 4-1BB polypeptide is the extracellular domain of mouse 4-1BB having the following sequence:

23 VQNSCDN CQPGTFCRKY NPVCKSCPPS

51 TFSSIGGQPN CNICRVCAGY FRFKKFCSST HNAECECIEG FHCLGPQCTR

101 CEKDCRPGQE LTKQGCKTCS LGTFNDQNGT GVCRPWTNCS LDGRSVLKTG

151 TTEKDVVCGP PVVSFSPSTT ISVTPEGGPG GHSLQV (SEQ ID NO: 7)

Another example of a soluble 4-1BB polypeptide is the extracellular domain of human 4-1BB having the following sequence:

SLQDPCSN CPAGTFCDNN RNQICSPCPP

51 NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHLCGAGCS

101 MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG

151 TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSP (SEQ ID NO: 8)

Other examples of soluble 4-1BB polypeptides include the first 186 amino acids of mouse 4-1BB or the first 185 amino acids of human 4-1BB as set forth in SEQ ID NOS: 20 and 21, respectively.

1 MGNNCYNVVV IVLLLVGCEK VGAVQNSCDN CQPGTFCRKY NPVCKSCPPS

51 TFSSIGGQPN CNICRVCAGY FRFKKFCSST HNAECECIEG FHCLGPQCTR

101 CEKDCRPGQE LTKQGCKTCS LGTFNDQNGT GVCRPWTNCS LDGRSVLKTG

151 TTEKDVVCGP PVVSFSPSTT ISVTPEGGPG GHSLQV (SEQ ID NO: 20)

1 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP

51 NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHLCGAGCS

101 MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG

151 TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSP (SEQ ID NO: 21)

The soluble 4-1BB polypeptide can also have one or more additional amino acids at the N or C terminus of SEQ ID NO: 7, 8, 20 or 21, as long as the polypeptide can inhibit or reduce the activity of 4-1BBL, and if the polypeptide Produced by a cell, it does not bind to the cell's membrane as a transmembrane protein. For example, a soluble 4-1BB polypeptide can be fused to, for example, glutathione s-transferase (GST), the Fc region of human IgG, a histidine tag, or any of the unrelated amino acid sequences discussed above with SEQ ID NO: A fusion protein consisting of 7, 8, 20 or 21. The soluble 4-1BB polypeptide can also be a fragment of the extracellular domain, for example, a fragment of SEQ ID NO: 7, 8, 20 or 21, as long as the fragment has biological activity, that is, the fragment can inhibit or reduce the activity of 4-1BB. Methods for determining whether the fragment is biologically active, i.e., whether the fragment is capable of inhibiting or reducing 4-1BBL activity, include, but are not limited to, methods discussed and exemplified herein, for example, by assaying LPS-responsive cells such as macrophages ), and the 4-1BB, soluble 4-1BB, and biologically active fragments thereof present may be from any source. For example, they can be isolated from mice, rabbits, pigs, dogs, cows, monkeys or humans. They can be expressed from recombinant nucleic acids of host cells (eg, CHO, S2 and E. coli) or host animals (eg, pig or monkey or rabbit), and then isolated using protein purification techniques known to those skilled in the art. Alternatively, they can be synthesized by conventional peptide synthesis methods. Soluble 4-1BB can be derived from a peptidase digested preparation of the full-length polypeptide. Methods of making fusion proteins (eg, mouse 4-1BB-human Ig-Fc polypeptides) are well known in the art and include, for example, methods involving recombinant DNA techniques. More specifically, the nucleic acid sequence encoding the relevant 4-1BB domain can be linked to the nucleic acid encoding the Fc fragment and then expressed in an appropriate host cell. A number of expression vectors encoding fusion moieties into which nucleic acids encoding 4-1BB or biologically active fragments thereof can be cloned are already commercially available. The soluble 4-1BB used was purchased from a company.

4-1BBL-specific antisense RNA

Antisense RNA can be used to specifically reduce the expression of 4-1BBL by, for example, inhibiting transcription and/or translation. The antisense RNA is complementary to the sense nucleic acid encoding 4-1BBL. For example, it can be complementary to the coding strand of a double-stranded cDNA molecule, or it can be complementary to an mRNA sequence. It can be complementary to the entire coding strand or only a portion thereof. It can also be complementary to all or part of the non-coding region of the nucleic acid encoding 4-1BBL. The noncoding region includes 5' and 3' regions flanking the coding region, eg, 5' and 3' untranslated sequences. Antisense RNAs are at least six nucleotides in length, but can be about 8, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. Longer oligonucleotides can be used. Antisense RNA can be prepared by methods known in the art, for example, by expression from an expression vector encoding the antisense RNA or from an expression cassette. Alternatively, it can be prepared using non-naturally occurring nucleotides or modified nucleotides designed to increase the biological stability of the RNA or to increase the interaction between the antisense RNA and the sense nucleic acid. Physiological stability of the duplex formed. Examples of modified nucleotides include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl ) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosyl Q nucleoside, inosine, N6-prenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylguanine Cytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosyl Q nucleoside, S′-methoxycarboxymethyl 1-uracil, 5-methoxyuracil, 2-methylthio-N6-prenyl adenine, uracil-5- Oxyacetic acid, wybutoxosine, pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, urine Pyrimidine-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3 )w, and 2,6-diaminopurine. Therefore, the antisense RNA of the present invention may include modified nucleotides and natural nucleotides, and it may have any length described above that is complementary to the sequences provided in SEQ ID NOs: 3 and 4.

4-1BBL-specific siRNA

Small interfering RNA can be used to specifically reduce 4-1BBL translation, thereby reducing 4-1BBL polypeptide levels. siRNA can mediate post-transcriptional gene silencing in a sequence-specific manner. See, eg, http://www.ambion.com/techlib/hottopics/rnai/rnai_may2002_print.html (last retrieved May 10, 2006). Typically integrated into the RNA-induced silencing complex, siRNA mediates cleavage of the cognate endogenous mRNA transcript by directing the complex to the cognate mRNA transcript, which is cleaved by the complex. The siRNA can be homologous to any region of the G protein mRNA transcript. The region of homology may be 30 nucleotides or less in length, preferably less than 25 nucleotides, more preferably about 21 to 23 nucleotides in length. SiRNAs are usually double-stranded and may have two nucleotides 3'overhang, eg, a 3'overhang UU dinucleotide. Methods for designing siRNAs are known to those skilled in the art. See, eg, Elbashir et al. Nature 411:494-498 (2001); Harborth et al. Antisense Nucleic Acid Drug Dev. 13:83-106 (2003). Typically selections start with AA, have 3' UU overhangs on both the sense and antisense siRNA strands, and target sites with approximately 50% G/C content. SiRNA can be chemically synthesized, produced by internal and external transcription, or expressed by siRNA expression vectors or PCR expression cassettes. See, eg, http://www.ambion.com/techlib/tb/tb_506.html (last retrieved May 10, 2006). When an siRNA is expressed from an expression vector or a PCR expression cassette, the insert encoding the siRNA can be expressed as an RNA transcript folded into an siRNA hairpin. Thus, the RNA transcript may comprise a sense siRNA sequence linked by a spacer sequence to its anticomplementary antisense siRNA sequence to form the loop of the hairpin and a U-string at the 3' end. The loop of the hairpin may be of any suitable length, For example, 3 to 30 nucleotides, preferably, 3 to 23 nucleotides, and can have various nucleotide sequences, including AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC and UUCAAGAGA (SEQ ID NO: 9). SiRNAs can also be produced by in vivo cleavage of double-stranded RNAs that are introduced directly or through transgenes or viruses. Amplification by RNA-dependent RNA polymerase can occur in some organisms.

Exemplary siRNA sequences that hybridize to 4-1BBL mRNA transcripts include the following and their complements:

SEQ ID NO. sequence

10 AAGACGGCGCAGGCGGCAGCG

11 AAGAGGCCGGCGGGATCGTCG

12 ATAGTAGACTCCAGCCTTGGC

13 ATTCCGACCTCGGTGAAGGG

22 AAGAGGCCGGCGGGAUCGUCG

23 AUAGUAGACUCCAGCCUUGGC

24 AUUCCGACCUCGGUGAAGGG

Their complementary sequences are:

25 CGCTGCCGCCTGCGCCGTCTT

26 CGACGATCCCGCCGGCCTCTT

27 GCCAAGGCTGGAGTCTACTAT

28 CCCTTCACCGAGGTCGGAAT

29 CGCUGCCGCCUGCGCCGUCUU

30 CGACGAUCCCGCCGGCCUCUU

31 GCCAAGGCUGGAGUCUACUAU

32 CCCUUCACCGAGGUCGGAAU

Small interfering RNAs targeting mouse 4-1BBL also include:

5'-GCTCTATGGCCTAGTCGCT-3' (SEQ ID NO: 14);

5'-GCAAAGCCTCAGGTAGATG-3' (SEQ ID NO: 15)

5'-GCUCUAUGGCCUAGUCGCU-3' (SEQ ID NO: 33)

5'-GCAAAGCCUCAGGUAGAUG-3' (SEQ ID NO: 34)

Their complementary sequences are:

AGCGACTAGGCCATAGAGC (SEQ ID NO: 35)

CATCTACCTGAGGCTTTGC (SEQ ID NO: 36)

AGCGACUAGGCCAUAGAGC (SEQ ID NO: 37)

CAUCUACCUGAGGCUUUGC (SEQ ID NO: 38)

Additional siRNA sequences of the present invention include SEQ ID NO: 18 and 19 and complementary sequences thereof, and the following sequences:

AAGACGGCGCAGGCGGCAGCGTT (SEQ ID NO: 45)

AAGAGGCCGGCGGGAUCGUCGTT (SEQ ID NO: 46)

AUAGUAGACUCCAGCCUUGGCTT (SEQ ID NO: 47)

AUUCCGACCUCGGUGAAGGGTT (SEQ ID NO: 48)

CGCUGCCGCCUGCGCCGUCUUTT (SEQ ID NO: 49)

CGACGAUCCCGCCGGCCUCUUTT (SEQ ID NO: 50)

GCCAAGGCUGGAGUCUACUAUTT (SEQ ID NO: 51)

CCCUUCACCGAGGUCGGAAUTT (SEQ ID NO: 52)

GCUCUAUGGCCUAGUCGCUTT (SEQ ID NO: 53);

GCAAAGCCUCAGGUAGAUGTT (SEQ ID NO: 54)

AGCGACUAGGCCAUAGAGCTT (SEQ ID NO: 55)

CAUCUACCUGAGGCUUUGCTT (SEQ ID NO: 56).

The siRNAs of the invention may also be chemically modified to increase their stability under physiological conditions (eg, in serum, in circulation or in mammalian cells). Chemically modified siRNAs of the invention include cholesterol-bound, lipid-enveloped, and antibody-linked siRNAs. Small interfering RNAs of the present invention also include small interfering RNAs that bind to other hydrophobic liquid molecules (e.g., for example, bind high-density lipoprotein to form lipophilic conjugates), or they have a partial phosphorothioate backbone and in sense or antisense 2'-O-methyl sugar modification on the chain. Lipophilic conjugates of siRNA include those incorporating saturated, alkyl chains such as stearoyl (C18) and behenyl (C22) and those incorporating lithocholic acid and oleamide mixtures (lithocholic acid- Oleyl, C43) combinations.

4-1BBL-specific ribozyme

The 4-1BBL blocker can also be a ribozyme. Ribozymes are catalytically active RNA molecules capable of cleaving single-stranded nucleic acids (eg, mRNAs with regions of homology). See, eg, Cech, Science 236: 1532-1539 (1987); Cech, Ann. Rev. Biochem. 59: 543-568 (1990); Cech, Curr. Opin. Struct. Biol. 2: 605-609 (1992) ; Couture and Stinchcomb, Trends Genet. 12:510-515 (1996). Ribozymes can be used to catalyze the cleavage of 4-1BBL mRNA transcripts, thereby inhibiting the translation of this mRNA. See, eg, US Patent 5,641,673. The ribozyme has specificity for 4-1BBL nucleic acid, and can be designed based on the nucleotide sequences of SEQ ID NO: 3 and 4. Methods for the design and construction of ribozymes capable of reverse cleaving RNA molecules in a highly sequence-specific manner have been developed and described in the art. For example, see. Haseloff et al., Nature 334:585-591 (1988). Ribozymes can be targeted to specific RNAs by engineering discrete "hybridizing" regions into the ribozyme. The hybridization region comprises a sequence complementary to the target RNA that supports the specific hybridization target of the ribozyme. See, eg, Gerlach et al., EP321,201. The target sequence may be a fragment of about 10, 12, 15, 20, or 50 contiguous nucleotides selected from the nucleotide sequence of SEQ ID NO: 3 or 4. Longer complementary sequences can be used to increase the affinity of the hybridizing sequence for the target. The hybridization and cleavage regions of a ribozyme can be integrally associated; thus, upon hybridization to the target RNA by the complementary region, the catalytic region of the ribozyme can cleave the target. Alternatively, mRNA encoding 4-1BBL can be used to select catalytic RNAs with specific ribonuclease activity from a collection of RNA molecules. See, eg, Bartel and Szostak, Science 261:1411-1418 (1993).

Other 4-1BBL blockers

4-1BBL blockers also include inhibitors of other molecules involved in the expression or activity of 4-1BBL, including oligonucleotides that reduce the expression of these molecules or inhibitors of the activity of these molecules themselves. Molecules involved in 4-1BBL expression or activity include, for example, NF-κB; CREB; C/EBP; TLRs including TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9; MyD88; TRIF; p38; Jnk; Mek; and TRAF6. Oligonucleotides that reduce the expression of these molecules are similar to the oligonucleotides described above for use as oligonucleotide-based 4-1BBL blockers. They include siRNAs (e.g., SEQ ID NOs: 18 and 19), antisense nucleic acids, or ribozymes directed against molecules involved in 4-1BBL expression or activity. Active inhibitors include, for example, the NF-κB inhibitor sulfasalazine, the proteasome inhibitor MG-132, the p38 inhibitor SB203580, the Jnk inhibitor SP600125, and the Mek inhibitor PD98059.

reduce inflammation

4-1BBL blockers can be used to prevent or treat inflammatory diseases. 4-1BBL blockade can reduce inflammation by reducing the overproduction of inflammatory cytokines (eg, TNF or IL-6). Accordingly, in one embodiment, the present invention provides a method of reducing the production of inflammatory cytokines in a mammal. The mammal may be a mammal suffering from or predisposed to suffering from an inflammatory disease.

Inflammatory diseases include, but are not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, psoriasis, ankylosing spondylitis, Crohn's disease, irritable bowel syndrome, systemic juvenile chronic arthritis, or osteoarthritis loose disease. Mammals with an inflammatory disease can be identified based on the known symptoms of the disease. For example, mammals with Crohn's disease or rheumatoid arthritis may display higher levels of pro-inflammatory cytokines (e.g., IL-1, IL-6, MCP-1 and TNF) generation level or duration. The amount of this increase can be, for example, 5%, 10%, 15%, 20% or more than 20%. Other symptoms of inflammatory disease include, but are not limited to, pathological inflammation and joint destruction. Mammals susceptible to inflammatory diseases can be described, for example, in Vyse & Todd, Cell 85: 31 1-18 (1996); Kinne et al. Arthritis Research 3: 319-330 (2001) and Rioux & Abbas, Nature 435: 584-9 (2005)) Identify the genetic abnormalities discussed in .

4-1BBL blockers can be administered by a variety of routes. Exemplary routes of administration include parenteral, intravenous, intradermal, subcutaneous, oral, inhalation, transdermal (topical), transmucosal, and rectal administration. Systemic administration can be by transmucosal or transdermal means. For example, polypeptide-based blocking agents may be administered by subcutaneous injection as needed or at the discretion of a physician. Administration can also be by IV infusion over several hours. Oligonucleotide-based 4-1BBL blockers (such as antisense or siRNA) can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by intravenous injection, topical application, or stereotaxic injection. See, eg, US Patent 5,328,470 and Chen et al., PNAS 91:3054 (1994). Thus, a 4-1BBL blocker can be administered to a subject by direct injection at the tissue site or by in situ generation. Alternatively, it can be modified to target selected cells and then administered systemically. For example, antisense molecules can be modified such that they bind receptors or antigens expressed on the surface of selected cells. Other small molecule blockers can be administered orally.

依法利珠单抗

依那西普(Enbrel)，英失利昔单抗氨甲蝶呤(Rheumatrex)，以及奥马佐单抗

The 4-1BBL blocker can be used alone or in combination with a second agent, e.g., a known anti-inflammatory agent, e.g., adalimumab

Efalizumab

Etanercept (Enbrel), Inbreximab methotrexate (Rheumatrex), and omalizumab

The dose administered to a mammal can be any amount suitable for reducing 4-1BBL expression or activity. The dose may be an effective dose or an appropriate fraction thereof. This will depend on individual patient parameters including age, physical condition, size, weight, the disease being treated, the severity of the disease and any concurrent therapy. Factors in determining appropriate dosages are well known to those of ordinary skill in the art and can be determined by routine experimentation. For example, standard chemical and biological assays can be employed, and physicochemical, toxicological and pharmacokinetic properties can be determined by mathematical modeling techniques known in the fields of chemistry, pharmacology and toxicology. Therapeutic utility and dosing regimens can be extrapolated from the results of such techniques and by using appropriate pharmacokinetic and/or pharmacodynamic models. The exact amount administered to a patient will be given by the attending physician's responsibility. Then, the 4-1BBL blocker can be administered orally or by injection at a dose of about 0.05 to about 2000 mg/kg, preferably about 1 to about 200 mg/kg based on the body weight of the mammal. Oligonucleotide-based blocking agents may be administered (eg, orally or by injection) at a dose of about 0.05 to 500 mg/kg, preferably 0.5 to 50 mg/kg, based on the body weight of the mammal. The dosage range for an adult is usually 4 to 40,000 mg/day, preferably 40 to 4,000 mg/day. Since some of the agents of the invention have a long-acting effect, they may advantageously be administered at an initial dose of 80 to 4,000 mg on the first day, followed by lower doses of 20 to 1,000 mg on subsequent days. A patient may also insist on a lower or tolerated dose for medical reasons, psychological reasons, or indeed any other reason. The effective amount of the second agent as a component of the pharmaceutical composition will follow the manufacturer's recommendation of the second agent, the judgment of the attending physician, and is subject to the amounts and dosages set forth in the Physician's Desk Reference (PHYSICIAN'S DESK REFERENCE) Guidance on regimens and administration factors.

Effectiveness of a method of treatment can be assessed by improvement in a patient's known signs or symptoms of disease. For example, amelioration of inflammation can be measured by monitoring the level and duration of pro-inflammatory cytokine production (eg, IL-1, IL-6, TNF or MCP-1 production). Any reduction in the level or duration of pro-inflammatory cytokine production, for example a reduction of 5%, 10%, 15%, 20% or more, is indicative of improvement in the patient.

Pharmaceutical composition of 4-1 blocker

A 4-1BBL blocker may be incorporated into a pharmaceutical composition suitable for administration to a mammal (referred to herein as a pharmaceutical composition of the invention). The pharmaceutical compositions of the present invention generally comprise an active agent and a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents known in the art to be compatible with pharmaceuticals administered to mammals. agent etc. Unless any conventional media or agents are incompatible with the active compounds, they can be used in the pharmaceutical compositions of the present invention. Furthermore, supplementary active compounds can also be included in the pharmaceutical compositions. For example, the pharmaceutical composition of the present invention may further include the above-mentioned second anti-inflammatory agent.

A therapeutically effective amount of a blocking agent may be employed in the compositions and methods of the invention. Such a therapeutically active amount of blocking agent is generally sufficient to reduce 4-1BBL expression or activity in the target cell or tissue or mammal. In some embodiments, the therapeutically effective amount of the blocking agent is an amount sufficient to reduce inflammation. An example of a therapeutically effective amount of a blocking agent is the dose described in the previous section. Accordingly, the 4-1BBL blocker may be administered at a dose of about 0.05 to about 2000 mg/kg, preferably about 1 to about 200 mg/kg based on the body weight of the mammal. The oligonucleotide-based blocking agent can be administered orally or by injection at a dose of about 0.05 to 500 mg/kg, preferably 0.5 to 50 mg/kg (eg 1, 3, 5 mg/kg) based on the body weight of the mammal. The dosage range for an adult is usually 4 to 40,000 mg/day, preferably 40 to 4,000 mg/day. Since some of the agents of the invention have a long-acting effect, they may advantageously be administered at an initial dose of 80 to 4,000 mg on the first day, followed by lower doses of 20 to 1,000 mg on subsequent days.

The pharmaceutical compositions of the invention may be formulated to suit the intended route of administration. Solutions or suspensions that can be used for parenteral, intradermal or subcutaneous application include (1) sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; (2) Antibacterial agents such as benzyl alcohol or methylparaben; (3) antioxidants such as ascorbic acid or sodium bisulfite; (4) chelating agents such as ethylenediaminetetraacetic acid; and (5) buffers such as ethyl salts, citrates, or phosphates, and tonicity-adjusting agents such as sodium chloride or dextrose. The pH can be adjusted by acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials.

Pharmaceutical compositions can also be formulated and administered according to the nature of the 4-1BBL blocker. Oligonucleotide-type 4-1BBL blockers (e.g., siRNA) can be delivered in an exemplary manner by a vector-based delivery system as described by Li and Rossi, Methods Mol. Biol. 309:261-72 (2005) , Song et al., Nat.Med.9:347-51 (2003); Soutschek et al., Nature 432:173-178 (2004); Morrissey et al., Nat.Biotechnol 23:1002-1007 (2005); Song et al. , Nat.Biotechnol.23:709-717 (2005); and high pressure intravenous injection of siRNA and chemically modified siRNA (including cholesterol-binding, lipid enveloped and antibody-conjugated siRNA). Oligonucleotide-type 4-1BBL blockers (e.g., siRNA and ribozymes) can also be found, for example, by Yano et al., In Vivo Antitumor Activity of a New Cationic Liposome siRNA Complex, in NON-VIRAL GENETHERAPY, Springer Tokyo, 2005 The cationic liposomes described above are used for delivery.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile aqueous solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water or phosphate buffered saline. Compositions must be sterile and stable under the conditions of manufacture and storage and should be preserved against the contamination of microorganisms (eg, bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be achieved, for example, by the use of coatings such as lecithin, by maintaining the desired particle size in the dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by the use of various antibacterial and antifungal agents (eg, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal). Other ingredients such as isotonic or absorption delaying agents (for example, aluminum monostearate and gelatin) may also be included.

Sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with one or more of the ingredients discussed above, as required, and filtering through sterile filtration. Dispersions are obtained by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying to obtain a powder containing the active ingredient plus any additional desired ingredient from a sterile-filtered solution thereof.

Oral compositions may contain an inert diluent or an edible carrier. They can be enclosed in gel capsules or compressed into tablets. For oral therapeutic administration, the active compound may be combined with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using liquid carriers. Pharmaceutically compatible binding agents and/or auxiliary substances may also be part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: binding agents, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose; disintegrants, such as , alginic acid or corn starch; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylic acid, or orange Flavoring agent.

For administration by inhalation, the compositions can be delivered as an aerosol spray from a pressurized container or dispenser containing a suitable propellant (eg, a gas such as carbon dioxide) or a nebulizer.

For transmucosal or transdermal administration, penetrants known in the art to be suitable for the barrier to be penetrated may be used. These include cleansers, bile salts and fusidic acid derivatives for transmucosal administration, such as by nasal spray. For transdermal administration, the active compounds are formulated into ointments, ointments, gels or creams as known in the art.

In one embodiment, the agents of the invention are prepared with carriers that will protect the agent against rapid elimination from the body, such as a controlled release formulation including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers may also be used. These include ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Lipid suspensions that can be targeted to infected cells by monoclonal antibodies directed against viral antigens can also be used as pharmaceutically acceptable carriers. They can be prepared by methods known in the art.

Oral or parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of administration. The phrase "dosage unit form" refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for dosage unit forms depends upon the particular properties of the active compound and the particular therapeutic effect to be achieved.

Pharmaceutical formulations of the gene therapy vector may comprise the gene therapy vector in an acceptable diluent, or may comprise a sustained release matrix in which the gene delivery vector is embedded.

The pharmaceutical compositions of the present invention may be contained in a container, pack or dispenser together with instructions for administration. Such articles of manufacture will include instructions for the use of 4-1BBL blockers in the treatment of persistent inflammation. Additionally, the article of manufacture may also include instructions for use of the second agent for treating inflammation.

screening assay

The present invention also includes methods of screening for novel 4-1BBL blockers. Novel 4-1BBL blockers can be identified based on their ability to interfere with 4-1BBL oligomerization, 4-1BBL interaction with other signaling molecules (such as TRAF6 or TLRs), or 4-1BBL translocation to the cell membrane.

4-1BBL blockers capable of preventing oligomerization of 4-1BBL can be identified in cell-based assays for the presence or absence of candidate agents. Candidate agents whose presence mediates the binding of two 4-1BBLs are 4-1BBL blockers capable of preventing oligomerization of 4-1BBL (ie, the formation of aggregates of three or more 4-1BBLs). In general, the binding of two 4-1BBLs can be determined by standard methods including, but not limited to, the yeast two-hybrid system (see Fields and Song, Nature 340:245-46 (1989)) and protein fragment complementation assays (See http://www.abrf.org/JBT/Articles/JBT0012/jbt0012.html, last retrieved September 8, 2006), eg, β-lactamase complementation assay. In the yeast two-hybrid system, two 4-1BBL fusion proteins can be expressed in yeast cells. One fusion protein consisted of 4-1BBL fused to a DNA-binding domain and the other fusion protein consisted of 4-1BBL fused to a transcriptional activation domain. Binding of 4-1BBL results in the activation of a reporter gene that allows yeast to grow on media of known composition. In a β-lactamase complementation assay, _two β-lactamase fragments, Bla(a) and Bla(b) consisting _of amino acids 26-196 and 198-290, respectively, can be combined with 4 -1BBL fusion. Cells transfected with 4-1BBL-Bla (a) and 4-1BBL-Bla (b) were loaded with CCF2/AM. CCF2/AM diffuses through the cell membrane, and cytoplasmic esterases hydrolyze its ester function, releasing the β-lactamase substrate CCF2. Excitation of the coumarin donor in CCF2 at 409 nm directs FRET to the fluorescein acceptor, resulting in green fluorescence emission at 520 nm. The combination of two 4-1BBLs brings together the two β-lactamase fragments to form a β-lactamase. This β-lactamase further hydrolyzes CCF2 and separates the donor and acceptor, resulting in FRET breakdown. As a result, the isolated coumarin donor emitted blue fluorescence at 447 nm. This can be observed under a microscope, or detected by flow cytometry.

4-1BBL blockers capable of inhibiting or reducing the interaction of 4-1BBL with signal transduction molecules can be identified by cell-based methods. An example of such an assay is described here. In a cell-based method for identifying 4-1BBL blockers capable of inhibiting or reducing the interaction of 4-1BBL with signal transduction molecules (eg, TLR or TRAF6), candidate agents are employed and then assayed for whether 4-1BBL interacts with The signaling molecule interacts. Alternatively, agents that inhibit the interaction of 4-1BBL with TLRs or TRAF6 can be identified by cell-based screens similar to those described above for 4-1BBL aggregation. For example, 4-1BBL-yeast DNA-binding protein and TRAF6-yeast transcriptional activation domain, or TRAF6-yeast DNA-binding protein and 4-1BBL-yeast transcriptional activation domain can be used in the yeast two-hybrid system described above. Similarly, fusion proteins consisting of 4-1BBL-Bla(a) and TRAF6-Bla(b) or TRAF 6-Bla(a) and 4-1BBL-Bla(b) can be used for 4-1BBL and Determination of TRAF6 interaction.

4-1BBL blocking agents capable of inhibiting or reducing translocation of 4-1BBL to the cell surface can be identified by contacting cells expressing 4-1BBL with a selected agent and determining whether the 4-1BBL localizes to the cell membrane. Such cells may be mammalian cells, such as the RAW264.7 cell line.

4-1BBL blockers can also be identified by cell-based or animal models of inflammation. An example of a cell-based assay is described here. For example, cells that express an inflammatory cytokine (eg, TNF, IL-6, IL-1 or MCP-1) in response to an appropriate stimulus (eg, exposure to LPS) are contacted with a candidate agent. The production of inflammatory cytokines by cells exposed to the candidate agent is compared to cells not exposed to the candidate agent. For cell-based assays, any cell capable of producing inflammatory cytokines in response to an inflammation-inducing stimulus (eg, LPS) can be used. These cells include, for example, RAW264.7 cells, primary murine macrophages, and primary human monocytes. Similarly, 4-1BBL blockers can be signed by animal models of inflammation. For example, a candidate agent can be administered to a mammal, the production of inflammatory cytokines (TNF or IL-6) or other observable phenotypes associated with excessive persistent inflammation (such as increased body temperature or joint swelling) can be measured, and compared with unadministered The class of candidate agents is compared in animals.

The present invention will be further described by the following examples, which do not limit the scope of the invention described in the claims.

Example

Example 1: Materials and methods

Mice and reagents. TLR2-, TLR4-, MyD88-, TRIF (LPS2), and TRIF- and MyD88-deficient mice are described in Hoebe et al., Nature 424:743-48 (2003) and Kim et al., J. Exp. Med. 197: 1441-52 (2003). 4-1BBL-deficient mice were backcrossed eight times with C57BL/6 mice as described by DeBenedette et al., J. Immunol. 163:4833-41 (1999). 4-1BBL-deficient mice were backcrossed at least seven times with C57BL/6 mice as described by Vinay, J. Immunol. 173:4218-29 (2004). Sex and age-matched mice were used in the same experiments.

Kim et al., J. Exp. Med. 197: 1441-52 (2003) describe the preparation and culture of peritoneal macrophage RAW264.7 and 293T cells. Sex- and age-matched 4-1BBL-deficient and wild-type (WT) mice were injected intraperitoneally with LPS. Collect serum samples or monitor surviving individuals. The protocol for the use of animals was approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.

The following reagents were used: Pam ₃ CysSer(Lys) ₄ (EMC microcollections GmbH); LPS from E. coli O111:B4 (List Biological Laboratories); phosphorothioate-stabilized CpG DNA and R-848 (Invivogen); Poly I:C (Amersham Biosciences); Peptidoglycan (Fluka); IL-1β and TNF (R&D systems); Recombinant mouse 4-1BB-Fc and anti-4-1BBL (R&D systems); Cloned antibodies (clone TKS-I, e-Bioscience); anti-Flag (Sigma); anti-GFP (Clontech); antibodies against HA, Myc, MyD88, TLR4 and TRAF6 (Santa Cruz Biotechnology); Alexa Fluor 594-conjugated Donkey anti-goat IgG (Molecular Probes); anti-AU-1 (Covance); anti-GAPDH (Chemicon); Brefeldin A (Biolegend); and sulfasalazine, SB203580, SP600125 and PD98059 (Calbiochem ).

Plasmids, recombinant adenoviruses and PCR. Mammalian expression vectors can be constructed from full-length, extracellular domain-deleted (ΔExt) or cytoplasmic-domain-deleted (ΔCyt) human 4-1BBL cDNA, or full-length, pro712his mutated, extracellular domain-deleted (ΔExt ) or cytoplasmic domain deletion (ΔCyt) of human TLR4 cDNA to construct mammalian expression vectors. These can be fused to GFP (in pEGFP-Cl), Flag (in pcDNA3) or HA (in pcDNA3). Replication-defective adenovirus-5-expressing Mouse 4-1BBL (Adv-4-1BBL) or non-transgenic control (Adv-control).

Semi-quantitative PCR was performed as described by Kim et al., J. Exp. Med. 197:1441-52 (2003). For real-time PCR, 0.5 μg of total RNA was used to prepare cDNA with oligo(dT) ₁₂ as a primer. The SYBR green PCRMaster Mix kit (Applied Biosystems) was used in the real-time PCR analysis.

The full-length, cytoplasmic domain, extracellular domain deletion (ΔExt), or cytoplasmic domain deletion (ΔCyt) human 4-1BBL cDNA was cloned into fusion GFP (pEGFP-Cl), flag (in pcDNA3), or Mammalian expression vector for HA (pcDNA3). Using the same method described by Bukczynski et al., proc. Transgenic control (Adv-control). The oligonucleotides were cloned into p-suppressor to express expression against mouse 4-1BBL (#1,5'-GCTCTATGGCCTAGTCGCT-S' (SEQ ID NO: 14); #2,5'-GCAAAGCCTCAGGTAGATG-S' (SEQ ID NO: 15)), mouse 4-1BB (#1, 5'-ACCTGTAGCTTGGGAACATTT-S' (SEQ ID NO: 16); #2, 5'-AATACAATCCAGTCTGCAAGA-S' (SEQ ID NO: 17)), Or empty siRNA strand from control. The human IL3R, TLR 2, 3, 4 or 9 cDNA strands were cloned into the pcDNA3-flag expression vector.

transfection. Using Lipofectamine 2000 and referring to the manufacturer's instructions (Invitrogen), 293T cells were transfected with the expression vector, and cell lysates were prepared 24 hours later. For siRNA transfection, polyamine (Ambion) was used to target mouse TRAF6 (#1, 5'-GGAUCAUCAAGUACAUUGUTT-S' (SEQ ID NO: 18); #2, 5'-GGGCUACGAUGUGG AGUUUTT-3' (SEQ ID NO : 19); pre-designed siRNA (Ambion) or GFP as a control transiently transfected the mouse macrophage cell line RAW264.7. After 2 days of transfection, the cells were incubated with stimulators. Cell lysis was prepared after 24 hours Products or culture suspensions were used to analyze TRAF6 inhibition by Western blot or TNF-α production by ELISA.

Electrophoretic mobility shift, nuclear transcription activity and yeast two-hybrid assays. Nuclear extracts were prepared according to the previous protocol. Specific oligonucleotides are radiolabeled by [γ- ^32P ]. ATP and EMSA were performed according to the recommendations of the Promega protocol. TNF transcription rate was determined with reference to the transcriptional activity assay described by Han et al., Biochim. Biophys. Acta 1090:22-28 (1991). Two-hybrid screening was performed using human fetal brain and spleen cDNA libraries as described by Han et al., Nature 386:296-299 (1997). A portion of human TLR4 spanning the intracellular protein domain (amino acids 653-839) was subcloned into pAS2 vector for use as bait. 5 x 10 transformants were screened.

4-1BBL or 4-1BB-inhibits the generation of stable cell lines. RAW 264.7 cells were stably transfected with p-suppressor-4-1BBL, 4-1BB, or empty vector. For cell selection, cells were incubated in medium containing 500 μg/mL G418 for 2 weeks and then pooled. Cells were maintained in media containing G418.

Flow Cytometry. Peritoneal macrophages were suspended in PBS, 5% FCS, 0.01% sodium azide (FACS buffer) on ice. Surface 4-1BBL expression was assessed using a phycoerythrin-conjugated 4-1BBL-specific antibody (clone TKS-I, eBioscience). Total 4-1BBL expression can be assessed after fixation and permeabilization in fixation fixation-permeabilization buffer (Biolegend). Cells were stained with phycoerythrin-conjugated hFACS antibody in permeabilization buffer and washed with FACS buffer. Phycoerythrin-conjugated rat IgG2a, κ served as an isotype control.

Immunohistochemistry and immunoassay. Peritoneal macrophages were seeded on coverslips, fixed with 4% paraformaldehyde, and permeabilized by incubation in PBS containing 0.5% Triton X-100. After blocking with 2% BSA, cells were incubated with anti-mouse 4-1BBL followed by Alexa Fluor 594-conjugated donkey anti-goat IgG. Fluorescent images were visualized using AxioVision 3.1 software (Carl Zeiss, Inc.). The supernatant of macrophage culture or mouse serum was collected, and the concentration of TNF or IL-6 was detected by enzyme-linked immunosorbent assay (ELISA) kit (eBioscience) according to the manufacturer's protocol. Antibodies used for immunoprecipitation and immunoblotting procedures are indicated in the legend. The experimental steps can be referred to Zhou et al., Mol. Cell. Biol. 26:3824-34 (2006).

Measurement of cytokines. The supernatant of macrophage culture or mouse serum was collected, and the concentration of TNF-α or IL-6 was detected by enzyme-linked immunosorbent assay (ELISA) kit (eBioscience) according to the manufacturer's protocol.

Statistical analysis. Kaplan-Meier plots were drawn and differences in mouse survival were determined using the log-rank (log-rank) test. The statistical significance of differences in serum TNF was determined by Student's t-test.

Example 2: 4-1BBL is a TLR4-interacting protein

Yeast two-hybrid screening was used to identify proteins interacting with the intracellular domain of TLR4. Combine pAS2-TLR4(653-839aa) with 4-1BBL(5-254aa) (clone 1), 4-1BBL(3-254aa) (clone 2), p38((AF), p53 or nuclear fiber in pGAD10 vector Yeast cells were transfected with lamin. The "bait and prey" interaction was confirmed by growth of yeast on media lacking histidine. Two of the seven positive clones were 4-1BBL, cell surface type II trans Membrane protein (see Goodwin et al., Eur. J. Immunol. 23:2631-2641 (1993) and Alderson et al., Eur. J. Immunol. 24:2219-2227 (1994)). Contains TLR4 encoded in the bait vector The finding that yeast cells with 4-1BBL encoded in the intracellular domain and prey vector are able to grow in media lacking histidine due to interaction-mediated expression of the His3 reporter gene shows that: 4-1BBL, but not others An unrelated protein may interact with this TLR4 intracellular domain (Fig. 1A).

To confirm this interaction, HA-4-1BBL was expressed in 293 cells or co-expressed with flag-TLR4 or flag-IL-3 receptor (IL-3R), and its association was determined by co-immunoprecipitation. 293T cells were transfected with empty pcDNA3 vector (control) or 4-1BBL expression vector combined with empty pcDNA3 vector, flag-TLR4 or flag-IL-3R expression vector. Cells were lysed 24 hours after transfection. 2/3 of the cell lysates were immunoprecipitated with anti-HA antibody. Immunoblotting of lysates and immunoprecipitates with anti-flag and anti-HA antibodies showed that TLR4, but not IL-3R, was pulled down by 4-1BBL (Fig. 1B).

In order to characterize the interaction between 4-1BBL and TLR4, the following experiments were performed.

Use to express GFP-4-1BBL and Flag-TLR4, Flag-TLR4 deletion of the cytoplasmic domain (ΔCyt), Flag-TLR4 deletion of the extracellular domain (ΔExt), Flag-IL-3R, Myc-TLR4, or Myc- The plasmid of TLR4-P712H(Lps ^d ) was transfected into 293T cells, and the cells were used for immunoassay. Cell lysates were immunoprecipitated with anti-Flag and anti-Myc, and cell lysates and immunoprecipitates were immunoblotted with the indicated antibodies. The results showed that the TLR4 intracellular domain is involved in the interaction with 4-1BBL, however, mutations in the LR4 TIR domain (Lps ^d , Pro712H) had no substantial effect on the interaction between TLR4 and 4-1BBL (Fig. 1C) . Since the Lps ^d mutation disrupts the interaction between the TIR domain and MyD88, but not the structure of the TIR domain (see Xu et al., Nature 408:111-15 (2000)), different parts in the TLR4 TIR domain may Interacts with 4-1BBL and MyD88.

In addition, flag-TLR4 expression vectors were used to co-express GFP, GFP-4-1BBL, GFP-4-1BBL deletion of the cytoplasmic domain (ΔCyt), GFP-4-1BBL deletion of the extracellular domain (ΔExt), or GFP-4 Immunoassay was performed on 293T cells transfected with -1BBL only cytoplasmic domain (Cyt) vector. Cell lysates were immunoprecipitated with anti-flag antibody and immunoblotted with anti-flag and anti-GFP antibodies. The results show that for 4-1BBL to interact with TLR4, it must contain its cytoplasmic domain and it must attach to the cell membrane, since neither 4-1BBL without the cytoplasmic domain nor the 4-1BBL cytoplasmic domain alone interacts with TLR4 co-immunoprecipitated (Fig. 1D).

Example 3: 4-1BBL-deficient mice are more resistant to LPS-induced death

To determine the in vivo effects of 4-1BBL, 4-1BBL-deficient mice were analyzed and the results are as follows. Mice lacking 4-1BBL are viable, fertile, and healthy. These mice had normal lymphoid organoids but displayed a defect in CD8 ⁺ T cell memory of the virus. The numbers of peritoneal macrophages were comparable in wild-type and 4-1BBL-deficient mice (data not shown). Peritoneal injection of LPS into 4-1BBL-deficient and wild-type mice showed that 4-1BBL-deficient mice were more resistant to the lethal effects of LPS than wild-type mice (Fig. 2A). In particular, the lethal effect of LPS on wild-type mice was moderated by administration of anti-4-1BBL (Fig. 2B, C). The in vivo effects of 4-1BBL deletion may be due to loss of 4-1BBL-mediated TNF production in macrophages, and/or loss of activation of 4-1BB by 4-1BBL in cells of other lineages. Interestingly, mice lacking 4-1BB were also resistant to death induced by LPS and galactosamine injections, since they had fewer natural killer (NK) and NKT cells. However, the causes of LPS resistance in 4-1BBL-deficient and 4-1BB-deficient mice must be different because 4-1BBL-deficient mice contain normal numbers of NK and NKT cells (data not shown).

Example 4: 4-1BBL is involved in sustained TNF production

A. In Vivo Studies

To determine whether 4-1BBL is involved in TLR4-mediated host response, 4-1BBL knockout (KO) and wild-type mice were challenged with 0.5 mg dose of E. coli LPS as follows. 0.5 mg of LPS was injected intraperitoneally into 4-1BBL-/- and wild-type mice, and TNF levels were measured in serum collected at 2 and 6 hours.

The results showed that LPS-induced TNF production was greatly reduced in 4-1BBL KO mice compared to wild-type mice (Fig. 2D). In particular, LPS induced a rapid increase in serum TNF concentrations, which peaked 1 hour after LPS injection. Serum TNF concentrations in 4-1BBL-deficient mice were comparable to those in wild-type mice one hour after LPS injection and lower than those in wild-type mice at a later time (Fig. 2D). These data are consistent with the in vitro data presented below (Fig. 3A), where 4-1BBL deletion only affected TNF production at a later time after LPS treatment; however, TNF induction by LPS appeared to be faster in vivo than in vitro.

B. Studies Using Primary Macrophages

Since macrophages are the major source of TNF in LPS-challenged mice (Beutler et al. Nature 316:552-554 (1985)), peritoneal macrophages from 4-1BBL KO and wild-type mice were isolated and detected after LPS stimulation. TNF generation.

First, the effect of 4-1BBL on LPS-induced cytokine production in macrophages was determined by assaying cytokines in the medium of 4-1BBL-deficient and wild-type macrophages 24 hours after LPS stimulation. 4-1BBL-deficient macrophages produced less TNF, IL-6 and IL-12 than wild-type macrophages (Fig. 3A). IL-1β was unaffected or only slightly affected by 4-1BBL deletion (Fig. 3A). 4-1BBL-deficient macrophages produced wild-type amounts of IL-6 after 1L-1β stimulation, suggesting that 4-1BBL is selectively involved in LPS-induced cytokine responses (Fig. 3A).

In addition, TNF production at 6, 12 and 18 hours after LPS induction was also determined. Peritoneal macrophages from 4-1BBL-/- and wild-type mice were plated at ^2x106 /microwell in six-well plates and then treated with LPS (100 ng/mL). TNF levels in the cultures were measured at 6, 12 and 18 hours. Interestingly, TNF production in 4-1BBL KO macrophages was normal during the first 3 hours of LPS stimulation but almost completely ceased after this time point (Fig. The initiation of a further rise in TNF production later in the stimulus is critical.

4-1BBL was originally cloned as a ligand for 4-1BB, a T-cell surface receptor with costimulatory function (see Goodwin et al., Eur. J. Immunol 23:2631-2641 (1993)). Since 4-1BB is also expressed in macrophages, it was examined whether 4-1BB is involved in 4-1BBL-mediated TNF expression in LPS-treated cells. The following experiments were performed to determine whether 4-1BB is involved in LPS-induced TNF production.

Unexpectedly, 4-1BBL did not require the presence of 4-1BB to promote sustained LPS-induced TNF production, since the amount of LPS-induced TNF was similar to wild-type and 4-1BB-deficient macrophages ( Figure 3C). Thus, the 4-1BBL-4-1BB interaction either did not occur in macrophages or had no effect on sustained macrophage TNF production.

C. Studies Using RAW264.7 Macrophages

To determine whether 4-1BBL functions in the macrophage cell line RAW264.7 similarly to primary macrophages, 4-1BBL expression was inhibited using siRNA as follows. Briefly, RAW 264.7 cells stably transfected with p-suppressor-containing control siRNA or 4-1BBL-siRNA #1 or #2. Protein levels of 4-1BBL were detected by immunoblotting. Control and 4-1BBL suppressor cells treated with (1) LPS for 3, 9 and 24 hours and (2) treated with peptidoglycan (PG, 10 μg/ml), poly I:C (25 μg/ml), LPS (100 ng TNF production was detected in control and 4-1BBL-inhibited cells treated for 24 hours with CpG (5 μg/mL) or untreated medium (none). The results showed that siRNA effectively inhibited 4-1BBL in RAW264.7 cells (Figure 4A), and inhibited LPS- and other TLR ligands-induced TNF production (Figure 4B, C), thus positively 4-1BBL in RAW264.7 and primary macrophages have the same effect. 4-1BBL had no effect on LPS-induced NF-κB activation in RAW264.7 cells, as control and 4-1BBL treated with LPS (100 ng/mL) for 15, 30, 40, 50, 60 and 90 minutes inhibited RAW cells Comparable I-κB-α degradation was shown (Fig. 4D).

Then, 4-1BB expression in RAW264.7 cells was inhibited using siRNA as follows. RAW 264.7 cells stably transfected with p-suppressor-containing control siRNA, 4-1BBL-siRNA #1 or #2. 4-1BB mRNA was checked by RT-PCR. Control and 4-1BBL treated with LPS for 24 hours inhibited TNF production measured in cells. siRNA inhibited 4-1BB very efficiently in RAW264.7 cells, but this inhibition did not affect LPS-induced TNF production (Fig. 5A-B). 4-1BB is therefore necessarily involved in LPS-induced TNF production in macrophages and is therefore not linked to the condition of 4-1BBL in sustained TNF production. In conclusion, by inhibiting 4-1BBL and 4-1BB with siRNA, 4-1BBL in sustained TNF production in LPS-treated macrophages was also observed in the RA W264.7 macrophage cell line 4-1BB - dependent function (Fig. 4A-C and Fig. 5A-B).

D. Transcription Studies

To determine whether 4-1BBL affects TNF expression at the transcriptional and/or post-transcriptional stages, Tnf mRNA was examined in 4-1BBL-deficient and wild-type macrophages at different stages of LPS treatment (Fig. 3D). 4-1BBL-deficient and wild-type macrophages had similar amounts of peak TnfmRNA expression after 2 hours of LPS stimulation; however, the amount of TnfmRNA in 4-1BBL-deficient cells was much lower than that of wild-type at the later stage of LPS stimulation Macrophages (Fig. 3D). These data are consistent with the TNF protein data (Fig. 3B) and show that 4-1BBL affects the expression of Tnf mRNA transcripts.

To determine whether 4-1BBL affects the transcription of Tnf, nuclear post-transcriptional activity assays were performed. At 1 h after LPS stimulation, LPS-triggered Tnf transcription was observed in both wild-type and 4-1BBL-deficient macrophages; decline (Fig. 3E).

To ascertain that the lower Tnf mRNA levels found in the late period after LPS stimulation in 4-1BBL-deficient macrophages were also caused by changes in mRNA stability, we measured 4-1BBL-deficient and wild-type TnfmRNA half-life in macrophages. 4-1BBL deletion had a substantial effect on the stability of TnfmRNA (Fig. 3F). Thus, 4-1BBL affects both transcriptional and post-transcriptional regulation of TNF production.

Whether 4-1BBL deletion affects LPS-induced activation of NF-κB and other transcription factors can be determined by electrophoretic mobility shift assay (EMSA) as follows. 4-1BBL-/- and wild-type macrophages were stimulated with LPS for 1, 2, 4, 8 and 12 hours, and then collected for nuclear extract preparation. EMSA was performed using radiolabeled probes. The results showed that deletion of 4-1BBL had no effect on LPS-induced NF-κB activation (early response peaking at two hours) (Fig. 3G). This is consistent with the data in Figure 3B, showing that LPS-induced TNF production was not affected by 4-1BBL deletion during the first three hours. 4-1BBL deletion had little or no effect on LPS-induced AP-1 activity (Fig. 3G). In contrast, LPS-induced CREB and C/EBP activity, which occurred 8 hours after LPS stimulation, was significantly inhibited by 4-1BBL deletion (Fig. 3G). This impaired CREB and C/EBP activity can be attributed to a defect in sustained TNF production in 4-1BBL-deficient macrophages.

In order to investigate LPS-induced signal transduction pathways, NF-κB and MAP kinase pathways, peritoneal macrophages from wild-type and 4-1BBL KO mice were treated with LPS (100 ng/mL) at 0.5, 1, 2, 4 and 6 hours, with anti-IκB-α, anti-phospho-ERK (p-ERK), anti-phospho-JNK (p-JNK), anti-phospho-p38 (p-p38), anti-4-1BBL, or anti - GAPDH antibody for immunoblotting of cell lysates. The results showed that the degradation rates of NF-kB (IκB-α) inhibitors were the same in 4-1BBL KO and wild-type cells (Fig. 3H), while ERK1/2, JNK1/2, and p38MAP kinases in 4-1BBL KO cells The level of activation (phosphorylation) was equal to or slightly lower than that of wild-type cells (Fig. 3H).

Thus, early downstream signaling of TLR4 appears to be unaffected by loss of 4-1BBL, and data obtained from analyzes of signal transduction pathways (Fig. 3H) and activation of transcription factors (Fig. 3G) are consistent with those obtained from analyzes of TNF production ( Figure 3B). Furthermore, it supports the notion that early signaling by TLR4 is intact in 4-1BBL knockout macrophages.

Example 5: 4-1BBL interacts with TLR2, TLR3, TLR4 and TLR9 and is involved in TLR2-, TLR3-, TLR4- and TLR9-mediated TNF production in macrophages

To determine whether 4-1BBL is selectively involved in TLR4-mediated cellular responses, HA-4-1BBL was co-expressed with flag-TLR2, flag-TLR3, flag-TLR4 or flag-TLR9 as follows. 293T cells were transfected with empty (control) or HA-4-1BBL expression vector combined with flag-TLR2, flag-TLR3, flag-TLR4, or flag-TLR9. Cells were lysed 24 hours after transfection. Cell lysates were immunoblotted with anti-flag antibody, immunoprecipitated with anti-HA followed by anti-HA and anti-flag. The results showed that all tested TLRs were pulled down by 4-1BBL in the co-immunoprecipitation assay (Fig. 6A). This co-immunoprecipitation was specific because flag-IL-3R did not co-immunoprecipitate with 4-1BBL (Fig. 6A).

To determine whether 4-1BBL is involved in TLR2-, 3-, 4- or 9-mediated cellular responses, the following experiments can be performed. With Pam3 (1μg/mL), poly I:C (25μg/mL), LPS (100ng/mL), R848 (100nM), CpG (5μg/mL), IL-1β (10ng/mL) or untreated medium (None) treatment of wild-type and 4-1BBL-/- macrophages. TNF production was measured 24 hours after treatment. The results showed that Pam3 (TLR2 ligand)-, PolyI:C (TLR3 ligand)-, R848 (TLR7&8 ligand)-, CpG (TLR9 ligand)-induced TNF production was reduced in 4-1BBLKO cells (Figure 6B ). This effect was TLR specific, as IL-1β-induced TNF production in 4-1BBL KO cells was normal (Fig. 6B). Thus, 4-1BBL is involved in signal transduction by many, if not all, different TLRs.

Example 6: Sustained TNF production in LPS-treated macrophages requires 4-1BBL induction and cell surface localization

4-1BBL is undetectable in most tissues and is only expressed at low levels in macrophages and dendritic cells (see Futagawa et al., Int. Immunol. 14:275-286 (2002)). To analyze the role of 4-1BBL in the sustained TNF production in LPS-treated macrophages, macrophages were isolated from wild-type as well as TLR4-/-, MyD88-/- and TRIF-/- mice and treated with LPS Treatments were 0.5, 1, 2, 4 and 8 hours. 4-1BBL protein was analyzed by immunoblotting using anti-4-1BBL antibody. GAPDH was used as a loading control. The results showed that LPS induced a rapid expression of 4-1BBL in macrophages, with a peak at 4 hours (Fig. 7A, left lanes 1-5 or 1-6 in all figures). The timing of 4-1BBL induction correlated well with impaired sustained TNF production in 4-1BBL knockout macrophages (Fig. 3B). As LPS-induced 4-1BBL expression was impaired in TLR4-/-, MyD88-/- and TRIF-/- macrophages, it was TLR4-, MyD88- and TRIF-dependent (Fig. 7A). 4-1BBL expression was induced by all TLR ligands tested, but not by 1L-1β (Fig. 8A-F). It was shown that 4-1BBL was post-translationally modified (probably by glycosylation) after its synthesis, as shifted protein bands were observed on SDS-PAGE (Fig. 7A).

The promoter of the gene encoding 4-1BBL with multiple NF-κB-binding sites, and the LPS-induced 4-1BBL mRNA was inhibited by the NF-κB inhibitor sulfasalazine (Fig. 7B) or the proteasome inhibitor MG-132 (data not shown) inhibited efficiently. The p38 inhibitor SB203580, the Jnk inhibitor SP600125 and the Mek inhibitor PD98059 also had certain inhibitory effects on the expression of 4-1BBL (Fig. 7B). LPS-induced 4-1BBL mRNA (T _1/2 = 67min) expressed by adenoviral vectors was more stable than 4-1BBL mRNA (T _1/2 = 33min) in unstimulated macrophages, showing that LPS-induced LPS-induced changes in mRNA stability were also involved in the expression of 4-1BBL (Fig. 7C).

4-1BBL expression in LPS-treated wild-type mouse peritoneal macrophages was analyzed by flow cytometry using an anti-4-1BBL antibody. An isotype antibody was used as a control. Permeabilize with 0.1% saponin to detect total 4-1BBL. The results showed that the majority of LPS-induced 4-1BBL localized on the cell surface (Fig. 7D).

To determine whether newly synthesized 4-1BBL is required on the cell surface to regulate TNF expression, peritoneal macrophages were pre-incubated for 1 hr with brefeldin A (3 μg/mL) alone or in combination, and then treated with LPS for the indicated times. Immunostaining was performed using anti-4-1BBL antibody. 4-1BBL and TNF mRNA were analyzed by semi-quantitative PCR. GAPDH was used as a control. Brefeldin A is a specific inhibitor of protein translocation to the cell surface by inhibiting protein translocation from the endoplasmic reticulum to the Golgi apparatus (Klausner et al., J. Cell Biol. 116:1071-1080 (1992)) ( Figure 7E). Blocking the translocation of newly synthesized 4-1BBL to the cell surface did not affect the LPS-induced increase in 4-1BBL mRNA (Fig. 7F). Brefeldin A treatment did not affect LPS-induced TNF mRNA after 1 h, but reduced TNF mRNA at 2, 4, and 6 h, suggesting that not only 4-1BBL induction but also 4-1BBL induction is required for sustained TNF production localization on the cell surface.

Example 7: Expression and crosslinking of 4-1BBL triggers TNF production

Sustained TNF production by LPS-treated macrophages requires induction of 4-1BBL, it was examined whether overexpression of 4-1BBL triggers TNF production. 4-1BBL was expressed in macrophages by adenovirus-mediated gene delivery as described by Bukczynski et al., Proc. Natl. Acad. Sci. U.S.A. 101:1291-1296 (2004). Briefly, macrophages were infected with different doses of empty (control) or adenovirus encoding 4-1BBL, and the expression of 4-1BBL was determined by immunoblotting with anti-4-1BBL antibody. The TNF level in the medium was detected 24 hours after virus infection. The results showed that 4-1BBL expression induced TNF production in a dose-dependent manner (Fig. 9A). This TNF induction was specific for 4-1BBL since there was no detectable TNF in the medium of control virus-infected cells. TNF induction by 4-1BBL-expression does not require 4-1BB, as TNF production was similar in 4-1BBL-deficient and wild-type macrophages infected with adenovirus encoding 4-1BBL (Fig. 9B).

To test whether 4-1BBL overexpression-mediated TNF production is TLR4-, MyD88- and TRIF-dependent, TLR4-/- and TRIF-/- macrophages were infected with control or 4-1BBL expressing virus, and then the cells were detected 4-1BBL levels in the medium and TNF levels in the medium. The results showed that the mediation of TNF production by 4-1BBL expression in macrophages isolated from TLR4-/- mice was partially disrupted (Fig. 9C), suggesting that in addition to TLR4, other TLRs that can interact with 4-1BBL also Involved in 4-1BBL-mediated TNF production. Indeed, 4-1BBL could interact with TLR2 and other TLRs when co-expressed in 293T cells (Fig. 6A), and TNF production induced by 4-1BBL overexpression was reduced in TLR2-deficient macrophages (Fig. 9D). These results support the explanation that multiple TLRs are involved in 4-1BBL-mediated TNF production. Despite the involvement of TLRs, TRIF deficiency had no effect on 4-1BBL-induced TNF production (Fig. 9E). Since macrophages isolated from MyD88 mice were not efficiently infected by adenovirus, the requirement for MyD88 in 4-1BBL-induced TNF production needed to be determined by another method (see below).

4-1BBL is a member of the TNF superfamily (see Watts, T.H., Annu. Rev. Immunol. 23:23-68 (2005)). Like other members of this superfamily, it requires a trimeric form for its function (see Rabu et al., J. Biol. Chem. 280:41472-41481 (2005)). To test this possibility, treatment with 4-1BB-Fc chimera (homodimer of disulfide-linked 4-1BB), anti-Fc antibody, or both 4-1BB-Fc and anti-Fc For wild-type macrophages, TNF levels in the medium were detected. More specifically, with goat anti-human Fc antibody (anti-Fc, 1.5 μg/mL), mouse 4-1BB-human Ig-Fc domain fusion protein (4-1BB-Fc, 5 μg/mL), 4- 1BB-Fc and anti-Fc together, or untreated medium treated macrophages. TNF levels in the medium were measured after 24 hours. The results showed that 4-1BB-Fc could cross-link two 4-1BBL molecules without inducing TNF production (Fig. 9F). Further cross-linking of 4-1BB-Fc with anti-Fc antibody induced TNF production, but anti-Fc antibody alone had no effect (Fig. 9F). It has been shown that cross-linking of more than two 4-1BBL molecules is required to initiate TNF production. The requirement for 4-1BBL for crosslink-induced TNF production was confirmed by using 4-1BBL KO cells (Fig. 9F).

To determine whether MyD88 and TRIF are required for 4-1BBL signaling, MyD88-/- and TRIF-/- macrophages were treated with 4-1BB-Fc, anti-Fc antibody, and 4-1BB-Fc and anti-Fc antibody cells, and detect the TNF level in the culture medium. The results showed that 4-1BBL cross-linking mediated TNF production was MyD88- and TRIF-dependent (Fig. 9F). Conversely, there was a statistically significant decrease in TNF production triggered by 4-1BBL crosslinking in TLR2-deficient and TLR4-deficient macrophages compared with wild-type macrophages (Fig. 9F).

One interpretation of these data is that expression and subsequent oligomerization of 4-1BBL on the cell surface triggers MyD88- and TRIF-independent TNF production. Due to the interaction of TLR4 and other TLRs with 4-1BBL (Figures 1A, 1B, 1D and 6A), and due to the formation of dimers by TLRs (see Medzhitov et al., Nature 388:394-397 (1997)), 4-1BBL The reason that overexpression-mediated TNF production is partially dependent on TLR4 (Fig. 9C) may be due to 4-1BBL-TLR4 interaction leading to 4-1BBL cross-linking. This is supported by the finding that TNF production mediated by 4-1BBL overexpression was partially inhibited by deletion of TLR4 or TLR2 (Fig. 9C-F).

Since 4-1BB-Fc and anti-4-1BBL bind only two 4-1BBL molecules and their binding to 4-1BBL should inhibit 4-1BBL oligomerization or prevent 4-1BBL from interacting with other molecules such as TLRs, They are used to test this hypothesis. Macrophages were stimulated with LPS in the presence of 4-1BB-Fc or anti-4-1BBL antibody as follows. Macrophages were incubated with LPS, LPS and 4-1BB-Fc, LPS and 0, 2, or 5 μg/mL anti-4-1BBL antibody, or with untreated medium for 24 hours. The results showed that both 4-1BB-Fc and anti-4-1BBL antibody inhibited LPS-induced TNF production (Fig. 9G).

Since 4-1BBL is induced by a variety of TLR ligands (Figure 8A-F), TLR2 ligand (Pam3), TLR3 ligand (poly I:C), TLR4 ligand (LPS), TLR7, 8 ligand (R848) and TLR9 ligand (CpG) stimulated TNF production by 4-1BBL-deficient macrophages. A reduction in TNF production was observed (Fig. 6B). These data show that 4-1BBL is involved in TNF production mediated by different TLRs. Since TLRs are located on endosomes or endoplasmic reticulum (ER) (such as TLR9), it is unlikely to interact with 4-1BBL located in the plasma membrane, TLR9-induced 4-1BBL signal transduction may depend on the interaction of 4-1BBL with Interaction of other TLRs on the cell surface. Consistent with this, a small but statistically significant decrease in TLR9-induced TNF production was detected in TLR4-deficient macrophages when a low dose of CpG (1 μg/mL) was used (Fig. 6C–D) . However, in addition to the interaction between 4-1BBL and cell surface TLRs, this mechanism may also involve 4-1BBL signaling, since when the cells were stimulated with high doses of CpG, the decline caused by TLR4-depletion was not obvious.

Example 8: Two consecutive TLR4 complexes

Since 4-1BBL induction is dependent on the TLR/MyD88/TRIF pathway, 4-1BBL-mediated signal transduction is independent of MyD88 and TRIF, but is related to its interaction with TLR, see below for LPS-treated macrophages Whether there is a continuous TLR4 signaling complex was examined. Macrophages were treated with LPS for 0.5, 1, 2, 4, 8 and 12 hours. The total cell lysate (1) was immunoblotted with anti-4-1BBL, anti-TLR4 and anti-MyD88; (2) was immunoprecipitated with anti-TLR4 antibody and treated with anti-TLR4, anti-4-1BBL and -MyD88 by immunoblotting; (3) immunoprecipitation with anti-MyD88 antibody and immunoblotting with anti-MyD88, anti-TLR4 and anti-4-1BBL; or (4) immunoprecipitation with anti-4-1BBL antibody And immunoblotting was performed with anti-4-1BBL, anti-TLR4 and anti-MyD88. An isotype antibody was used as a negative control for all immunoprecipitations. Positive controls for immunoblotting against 4-1BBL or MyD88 are shown in boxes. The results showed that LPS induced 4-4BBL expression but had no effect on the protein levels of TLR4 or MyD88 over a 2 to 8 hour exposure time (Fig. 10A, cell lysates). TLR4 accompanies MeD88 within half an hour to 1 hour of LPS exposure, but then dissociates from it, whereas the TLR4-4-1BBL complex appears within 2 to 12 hours of exposure (FIG. 10A, IP: TLR4). The TLR4-MyD88 complex was confirmed by immunoprecipitation of MyD88 (Fig. 10A, IP: MyD88). Myd88 cannot pull down 4-1BBL, showing that MyD88 is not in the same TLR4 complex as 4-1BBL. Immunoprecipitation of 4-1BBL pulled down TLR4 but failed to pull down MyD88 in 2-12 hr long LPS-treated cells, confirming that MyD88 and 4-1BBL are not in the same complex (Figure 10A, IP: 4-1BBL ). Thus, two consecutive TLR4 complexes exist in LPS-treated macrophages, one MyD88 complex responsible for initiating cellular responses and the other 4-1BBL-TLR4 complex involved in sustained TNF production.

To confirm that MyD88 cannot interact with 4-1BBL, AU1-tagged MyD88 was overexpressed with flag-4-1BBL or flag-TLR4 as follows. 293T cells were transfected with MyD88-AU1 expression vector combined with flag-4-1BBL or flag-TLR4 expression vector. Cells were lysed 24 hours after transfection, and cell lysates were immunoprecipitated with anti-AU1 antibody, followed by immunoblotting with anti-Flag and anti-AU1 antibodies. The results showed that Flag-TLR4, but not flag-4-1BBL, was pulled down by MyD88 (Fig. 10B).

To find proteins that interact with 4-1BBL, the interaction of 4-1BBL with TRAF2 and TRAF6 was examined as follows. 293T cells were transfected with HA-4-1BBL expression vector combined with TRAF2-myc or TRAF6-myc expression vector. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-myc and anti-HA antibodies. The results showed that 4-1BBL interacted with TRAF6, but not TRAF2 (Fig. 10C).

Due to the interaction of 4-1BBL and TRAF6, it was examined whether TRAF6 is required for 4-1BBL-mediated TNF production. SiRNA can be used to inhibit TRAF6 in RAW264.7 macrophages see below. Macrophages were transfected with TRAF6-siRNA #1 or #2, or control siRNA (C), and TRAF6 protein levels were analyzed by immunoblotting against TRAF6. Incubate with anti-Fc, 4-1BB-Fc, 4-1BB-Fc and anti-Fc, LPS, poly I:C (25 μg/mL), IL-1β (10 ng/mL), or untreated medium After 24 hours, the TNF production level of control siRNA-, TRAF6-siRNA#1- or TRAF6-siRNA#2-transfected cells was detected. IL-6 levels were measured when cells were treated with medium, LPS, or TNF (10 ng/mL). All siRNAs effectively reduced TRAF6 protein levels in RAW264.7 cells (Fig. 10D). TRAF6-inhibited and control cells were treated with 4-1BB-Fc, anti-Fc antibody, or 4-1BB-Fc and anti-Fc antibody together, and TNF levels in the culture medium were detected. Inhibition of TRAF6 blocked 4-1BBL cross-linking mediated TNF production (Fig. 10D). As expected, inhibition of TRAF6 impaired LPs, poly I:C and IL-1β-induced cytokine production, but had no effect on TNF-induced IL-6 production (Fig. 10D).

To illustrate downstream events triggered by 4-1BBL, transcription factor activation was examined. Expression of 4-1BBL activated CREB and C/EBP, but not NF-κB (Fig. 10E), consistent with the observation that deletion of 4-1BBL had no effect on LPS-induced NF-κB activation but impaired CREB and C /EBP activation (Fig. 3F). Similarly, no degradation of IκBα was observed in cells overexpressing 4-1BBL (Fig. 10F). Expression of 4-1BBL triggered partial phosphorylation of p38, Jnk and Erk (Fig. 10F), and inhibition of p38, Jnk and Erk (but not NF-κB) reduced 4-1BBL-mediated TNF production (Fig. 10G), It is suggested that the MAP kinase pathway is involved in 4-1BBL-mediated TNF production. The 4-1BBL signaling pathway appears to affect TnfmRNA, as 4-1BBL deletion resulted in a decrease in late LPS-induced TnfmRNA transcription (Fig. 3E), whereas 4-1BBL overexpression increased TnfmRNA quantity (Fig. 10H). The half-life of Tnf mRNA was compared in cells overexpressing 4-1BBL and cells treated with LPS; Tnf transcripts showed similar half-lives (Fig. 10H). Since LPS is known to stabilize TnfmRNA and deletion of 4-1BBL reduces LPS-induced TnfmRNA stabilization (Fig. 3F), 4-1BBL appears to be a mediator of LPS-induced TnfmRNA stabilization. Taken together, the data presented here show that the priming activation of TNF expression and the sustained production of TNF in macrophages is regulated by early and late signal transduction pathways (Fig. 10I).

The expression of pro-inflammatory cytokines is tightly controlled by gene activation and inactivation mechanisms. The studies presented here show that the priming activation of TNF expression and the sustained production of TNF is regulated by early and late signal transduction pathways (Fig. 10I). The well-known signaling pathway TLR4/MyD88/TRIF is responsible for the initiation and early expression of inflammatory genes. 4-1BBL is an early induced protein and is induced only in the early stages of cell activation. The newly synthesized 4-1BBL translocates to the cell surface to initiate a new signaling phase for sustained TNF production. The interaction between 4-1BBL and TLRs appears to be involved in the formation of the second signaling phase, and the role of TLRs in this continuous signaling complex is likely to assist the oligomerization of 4-1BBL. The signaling mechanism of the 4-1BBL complex differs from initial TLR4 signaling because it is independent of MyD88 and TRIF. However, TRAF6 is required for both signaling phases.

The production of inflammatory cytokines is part of the inflammatory process, characterized by an initial period and a subsequent duration that is usually terminated by the breakdown of inflammatory triggers (see Triantafilou and Triantafilou, Trends Immunol. 23:301-304 (2002)). Malfunctions of inflammation regulation often occur at the end of a sustained inflammatory response, and prolonged sustained TNF production often accompanies the pathology of many inflammatory diseases (see Vassalli, P., Annu. Rev. Immunol. 10:411-452 (1992)). Thus, the identification of 4-1BBL as a key regulator in sustained TNF production provides new therapeutic targets for the development of novel therapeutic interventions in inflammatory diseases.

Example 9: The role of 4-1BBL in IL-6 induction

IL-6 is a cytokine involved in inflammation. Elevated expression of IL-6 is associated with the pathology of a variety of disease processes, including rheumatoid arthritis (RA), systemic juvenile chronic arthritis (JCA), osteoporosis, and psoriasis. To show that 4-1BBL is involved in IL-6 induction, the following experiments were performed. First, 0.5 mg of LPS was injected intraperitoneally into 4-1BBL-/- and wild-type mice. Serum was then collected at 0, 2 and 6 hours, and IL-6 levels were measured. The results showed that 4-1BBL-/- mice produced significantly less IL-6 than wild-type mice in response to LPS (Fig. 11A). When peritoneal macrophages from 4-1BBL-/- and wild-type mice were treated with LPS (100 ng/mL) and the IL-6 levels in the medium were measured at 3, 9 and 18 hours, the results confirmed that those from wild-type Macrophages from 4-1BBL-/- mice produced significantly more IL-6 than those from 4-1BBL-/- mice (Fig. 1 IB). These results were compared with those using Pam3 (1 μg/mL), poly I:C (25 μg/mL), LPS (100 ng/mL), R848 (100 nM), CpG (5 μg/mL), IL-1β (10 ng/mL), TNF-α (10ng/mL), or untreated medium (none) treated wild-type and 4-1BBL-/- macrophages 24 hours after the IL-6 production levels were compared. Wild-type macrophages treated with Pam3, poly I:C, LPS, R848 and CpG produced significantly more IL-6 than 4-1BBL-/- macrophages (Fig. 11C). In contrast, wild-type and 4-1BBL-/- macrophages produced comparable levels of IL-6.

When macrophages were incubated with LPS, LPS and 4-1BB-Fc, LPS and 0, 2, or 5 μg/mL anti-4-1BBL antibody, or untreated medium for 24 hours, then IL- At the 6 level, the results showed that macrophages incubated with LPS and 4-1BB-Fc produced significantly less IL-6 than macrophages incubated with LPS alone (Fig. 1 ID). Macrophages incubated with LPS and increasing concentrations of anti-4-1BBL antibody also showed decreased levels of IL-6 production (Fig. 1 ID).

To confirm the role of 4-1BBL in IL-6 production, RAW 264.7 cells were stably transfected with p-inhibitor-containing control siRNA or 4-1BBL-siRNA #1 or #2. The IL-6 production of 4-1BBL suppressed cells treated with LPS for 3, 9 and 24 hours was detected and compared with control cells. The results showed that RAW264.7 cells with suppressed 4-1BBL expression produced significantly less IL-6 than RAW264.7 cells transfected with control siRNA (Fig. 1 IE). These results were compared with those treated with peptidoglycan (PG, 10 μg/mL), poly I:C (25 μg/mL), LPS (100 ng/mL), CpG (5 μg/mL) or untreated medium (no) for 24 hours. The results of the control and 4-1BBL inhibited cells were consistent ( FIG. 11F ).

cited literature

Galanos, C, Freudenberg, M.A. and Reutter, W. Galactosamine-induced sensitization to the lethal effects of endotoxin. Proc. Natl. Acad. ScL USA 76, 5939-5943 (1979).

Beutler, B. Inferences, questions and possibilities in Toll-like receptor signaling. Nature 430, 257-263 (2004).

Cohen, J. The immunopathogenesis of sepsis. Nature 420, 885-891 (2002). 4.

Janeway, C.A., Jr. and Medzhitov, R. Innate immune recognition. Annu. Rev. Immunol. 20, 197-216 (2002).

Akira, S. and Takeda, K. Toll-like receptor signaling. Nat. Rev. Immunol. 4, 499-511 (2004).

Goodwin, R.G. et al. Molecular cloning of a ligand for the inducible T cell gene 4-1BB: a member of an emerging family of cytokine with homology to tumor necrosis factor. Eur.J.Immunol.23, 2631-2641 (1993) .

Alderson, M.R. et al. Molecular and biological characterization of human 4-1BB and its ligand. Eur. J. Immunol. 24, 2219-2227 (1994).

Beutler, B. et al. Identity of tumor necrosis factor and themacrophage-secreted factor cachectin. Nature 316, 552-554 (1985).

Watts, T.H. TNF/TNFR family members in costimulation of Tcell responses. Annu. Rev. Immunol. 23, 23-68 (2005).

Xu, Y. et al. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 408, 111-115 (2000).

Futagawa, T. et al. Expression and function of 4-1BB and 4-1BBlig and on murine dendritic cells. Int. Immunol.14, 275-286 (2002).

Klausner, R.D., Donaldson, J.G. and Lippincott-Schwartz, J. Brefeldin A: insights into the control of membrane traffic and organellestructure. J. Cell Biol. 116, 1071-1080 (1992).

Bukczynski, J., Wen, T., Ellefsen, K., Gauldie, J. and Watts, T.H. Costimulatory ligand 4-1BBL(CD137L) as an efficient adjuvant for human antiviral cytotoxic T cell responses.Proc.Natl.Acad.ScL U.S. A101, 1291-1296(2004).

Rabu, C. et al. Production of recombinant human trimeric CD137L (4-1BBL). Crosslinking is essential to its T cell co-stimulation activity. J. Biol. Chem. 280, 41472-41481 (2005).

Medzhitov, R., Preston-Hurlburt, P. and Janeway, C.A., Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394-397 (1997).

Triantafilou, M. and Triantafilou, K. Lipopolysaccharide recognition: CD 14, TLRs and the LPS-activation cluster. Trends Immunol. 23, 301-304 (2002).

Vassalli, P. The pathophysiology of tumor necrosis factors. Annu. Rev. Immunol. 10, 411-452 (1992).

DeBenedette, M.A. et al. Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in rus the cytotoxicT cell response to influenza vi J. Immunol. 163, 4833-4841 (1999).

Hoebe, K. et al. Identification of Lps2 as a key transducer of MyD88-independent TIR signaling. Nature 424, 743-748 (2003).

Kim, S.O., Ono, K., Tobias, P.S. and Han, J. Orphan nuclear receptor Nur77 is involved in caspase-independent macrophage cell death. J. Exp. Med. 197, 1441-1452 (2003).

Han, J., Jiang, Y., Li, Z., Kravchenko, V.V. and Ulevitch, R.T. Activation of the transcription factor MEF2C by the MAP kinasep38 in inflammation. Nature 386, 296-299 (1997).

Ge, B. et al. TAB1 beta (transforming growth factor-beta-activated protein kinase 1-binding protein lbeta), a novel splicing variant of TAB1 that interacts with p38alpha but not TAK1. J. Biol. Chem. 278, 2286-2293 (2003 ).

other implementations

All patents and publications referenced or referred to herein represent the level of skill of those skilled in the art to which this invention pertains, and each such referenced patent or publication is incorporated herein to the same extent as if it were incorporated herein, individually or in its entirety, as an refer to. Applicants reserve the right to incorporate into this specification all material and information from any such cited patents and publications.

The particular methods and compositions described herein represent preferred embodiments and are exemplary and not intended to limit the scope of the invention. Those skilled in the art can find other objects, aspects and embodiments after referring to the description, which are included in the spirit of the invention defined by the scope of the claims. It will be apparent to those skilled in the art that various substitutions or modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention exemplarily described herein may be suitably practiced in the absence of any element, limitation, not specifically disclosed herein as critical. The methods and processes exemplarily described herein may be suitably performed in different order of steps, and they are not necessarily limited to the order of steps indicated herein or in the claims. As used in this specification and claims, the singular forms "a", "an" and "the" include plural referents unless expressly stated otherwise. Thus, for example, "an antibody" includes plural quantities (eg, an antibody solution or a series of antibody preparations) of that antibody. In no event is the invention to be construed as limited to the particular examples or embodiments or methods disclosed herein. In no event shall this invention be construed as limited by any statement by any examiner or other officer or employee of the Patent and Trademark Office, unless such statement is expressly adopted by applicant in a written response, without qualification and reservation.

The terms and expressions used herein are used as terms of description and not of limitation, and the use of such terms and expressions does not exclude equivalent features or partial equivalent features to those shown or described, but it is recognized that in the present invention Various modifications are possible within the claimed scope. Therefore, it should be recognized that although the invention has been specifically disclosed in terms of preferred embodiments and optional features, modifications and alterations to the disclosed concepts can be effected by those skilled in the art and such modifications and alterations are considered to be set forth in the appended claims. within the scope of the disclosed invention.

The invention has been described broadly and generically herein. Smaller species and subgeneric groups falling within the scope of the general disclosure also form part of this invention. This includes the general description of the invention with a proviso or negative limitation removing any subject matter from the parent concept, whether or not the ex vivo material is explicitly recited therein. Other embodiments are within the following claims. Furthermore, for functions and parts described in the invention in terms of Markush groups, those educated in the art will recognize that the invention is also described in terms of any individual member or subgroup member of the Markush group.

sequence listing

<110> Scripps Research Institute

Kang Yongjun

Han Jiahuai

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Gly Leu Leu Ala Asp Ala Ala Leu Leu Ser Asp Thr Val Arg Pro Thr

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Asn Ala Ala Leu Pro Thr Asp Ala Ala Tyr Pro Ala Val Asn Val Arg

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Asp Arg Glu Ala Ala Trp Pro Pro Ala Leu Asn Phe Cys Ser Arg His

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Pro Lys Leu Tyr Gly Leu Val Ala Leu Val Leu Leu Leu Leu Ile Ala

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Ala Cys Val Pro Ile Phe Thr Arg Thr Glu Pro Arg Pro Ala Leu Thr

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Ile Thr Thr Ser Pro Asn Leu Gly Thr Arg Glu Asn Asn Ala Asp Gln

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Val Thr Pro Val Ser His Ile Gly Cys Pro Asn Thr Thr Gln Gln Gly

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Ser Pro Val Phe Ala Lys Leu Leu Ala Lys Asn Gln Ala Ser Leu Cys

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Asn Thr Thr Leu Asn Trp His Ser Gln Asp Gly Ala Gly Ser Ser Tyr

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Leu Ser Gln Gly Leu Arg Tyr Glu Glu Asp Lys Lys Glu Leu Val Val

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Asp Ser Pro Gly Leu Tyr Tyr Val Phe Leu Glu Leu Lys Leu Ser Pro

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Thr Phe Thr Asn Thr Gly His Lys Val Gln Gly Trp Val Ser Leu Val

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Leu Gln Ala Lys Pro Gln Val Asp Asp Phe Asp Asn Leu Ala Leu Thr

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Val Glu Leu Phe Pro Cys Ser Met Glu Asn Lys Leu Val Asp Arg Ser

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Trp Ser Gln Leu Leu Leu Leu Lys Ala Gly His Arg Leu Ser Val Gly

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Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val

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Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe

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Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser

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Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp

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Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val

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Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp

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ctgaggtacg aagaagacaa aaaggagttg gtggtagaca gtcccgggct ctactacgta 600

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ctgctcgctg ccgcctgcgc cgtcttcctc gcctgcccct gggccgtgtc cggggctcgc 180

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Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln

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Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro

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Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys

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Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr

65 70 75 80

His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro

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Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr

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Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn

115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg

130 135 140

Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro

145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu

165 170 175

Gly Gly Pro Gly Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala

180 185 190

Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe

195 200 205

Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln

210 215 220

Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser

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Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu

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<212>PRT

<213> Homo sapiens

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Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

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Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

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Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

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Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

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Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

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Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

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Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

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Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

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Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

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<213> Mus musculus

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Val Gln Asn Ser Cys Asp Asn Cys Gln Pro Gly Thr Phe Cys Arg Lys

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Tyr Asn Pro Val Cys Lys Ser Cys Pro Pro Ser Thr Phe Ser Ser Ile

20 25 30

Gly Gly Gln Pro Asn Cys Asn Ile Cys Arg Val Cys Ala Gly Tyr Phe

35 40 45

Arg Phe Lys Lys Phe Cys Ser Ser Thr His Asn Ala Glu Cys Glu Cys

50 55 60

Ile Glu Gly Phe His Cys Leu Gly Pro Gln Cys Thr Arg Cys Glu Lys

65 70 75 80

Asp Cys Arg Pro Gly Gln Glu Leu Thr Lys Gln Gly Cys Lys Thr Cys

85 90 95

Ser Leu Gly Thr Phe Asn Asp Gln Asn Gly Thr Gly Val Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Arg Ser Val Leu Lys Thr Gly Thr

115 120 125

Thr Glu Lys Asp Val Val Cys Gly Pro Pro Val Val Ser Phe Ser Pro

130 135 140

Ser Thr Thr Ile Ser Val Thr Pro Glu Gly Gly Pro Gly Gly His Ser

145 150 155 160

Leu Gln Val

<210>8

<211>163

<212>PRT

<213> Homo sapiens

<400>8

Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp

1 5 10 15

Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser

20 25 30

Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly

35 40 45

Val Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys

50 55 60

Asp Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys

65 70 75 80

Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys

85 90 95

Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg

100 105 110

Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly

115 120 125

Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser

130 135 140

Pro Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly

145 150 155 160

His Ser Pro

<210>9

<211>9

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>9

uucaagaga 9

<210>10

<211>21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>10

aagacggcgc aggcggcagc g 21

<210>11

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<223> Synthetic oligonucleotides

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<210>12

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<213> Artificial sequence

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<223> Synthetic oligonucleotides

<400>12

atagtagact ccagccttgg c 21

<210>13

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>13

attccgacct cggtgaaggg 20

<210>14

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>14

gctctatggc ctagtcgct 19

<210>15

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>15

gcaaagcctc aggtagatg 19

<210>16

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>16

acctgtagct tgggaacatt t 21

<210>17

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>17

aatacaatcc agtctgcaag a 21

<210>18

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>18

ggaucaucaa guacauugut t 21

<210>19

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>19

gggcuacgau guggaguuut t 21

<210>20

<211>186

<212>PRT

<213> Mus musculus

<400>20

Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val

1 5 10 15

Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln

20 25 30

Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro

35 40 45

Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys

50 55 60

Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr

65 70 75 80

His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro

85 90 95

Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr

100 105 110

Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn

115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg

130 135 140

Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro

145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu

165 170 175

Gly Gly Pro Gly Gly His Ser Leu Gln Val

180 185

<210>21

<211>185

<212>PRT

<213> Homo sapiens

<400>21

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro

180 185

<210>22

<211>21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>22

aagaggccgg cgggaucguc g 21

<210>23

<211>21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>23

auaguagacu ccagccuugg c 21

<210>24

<211>20

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>24

auuccgaccu cggugaaggg 20

<210>25

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>25

cgctgccgcc tgcgccgtct t 21

<210>26

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>26

cgacgatccc gccggcctct t 21

<210>27

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>27

gccaaggctg gagtctacta t 21

<210>28

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>28

cccttcaccg aggtcggaat 20

<210>29

<211>21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>29

cgcugccgcc ugcgccgucu u 21

<210>30

<211>21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>30

cgacgauccc gccggccucu u 21

<210>31

<211>21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>31

gccaaggcug gagucuacua u 21

<210>32

<211>20

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>32

cccuucaccg aggucggaau 20

<210>33

<211>19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>33

gcucuauggc cuagucgcu 19

<210>34

<211>19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>34

gcaaagccuc agguagaug 19

<210>35

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>35

agcgactagg ccatagagc 19

<210>36

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>36

catctacctg aggctttgc 19

<210>37

<211>19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>37

agcgacuagg ccauagagc 19

<210>38

<211>19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>38

caucuaccug aggcuuugc 19

<210>39

<211>21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>39

accuguagcu ugggaacauu u 21

<210>40

<211>20

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>40

aauacaaucc agucugcaag 20

<210>41

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>41

aaatgtccc aagctacagg t 21

<210>42

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>42

tcttgcagac tggattgtat t 21

<210>43

<211>21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>43

aaauguuccc aagcuacagg u 21

<210>44

<211>21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>44

ucuugcagac uggauuguau u 21

<210>45

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<400>45

aagacggcgc aggcggcagc gtt 23

<210>46

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>46

aagaggccgg cgggaucguc gtt 23

<210>47

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>47

auaguagacu ccagccuugg ctt 23

<210>48

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>48

auuccgaccu cggugaaggg tt 22

<210>49

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>49

cgcugccgcc ugcgccgucu utt 23

<210>50

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>50

cgacgauccc gccggccucu utt 23

<210>51

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>51

gccaaggcug gagucuacua utt 23

<210>52

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>52

cccuucaccg aggucggaau tt 22

<210>53

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>53

gcucuauggc cuagucgcut t 21

<210>54

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>54

gcaaagccuc agguagaugt t 21

<210>55

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>55

agcgacuagg ccauagagct t 21

<210>56

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>56

caucuaccug aggcuuugct t 21

<210>57

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>57

accuguagcu ugggaacauu utt 23

<210>58

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>58

aauacaaucc agucugcaag att 23

<210>59

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>59

aaauguuccc aagcuacagg utt 23

<210>60

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotides

<220>

<223> Combined DNA/RNA

<400>60

ucuugcagac uggauuguau utt 23

Claims (62)

1. the pharmaceutical composition that comprises 4-1BBL blocker and pharmaceutically acceptable carrier, wherein this 4-1BBL blocker is selected from the antagonistic antibodies of (a) specificity at 4-1BBL; (b) effective oligonucleotide that reduces the 4-1BBL expression of nucleic acid in cell; (c) solubility 4-1BB.

2. pharmaceutical composition as claimed in claim 1, wherein this antagonistic antibodies is the chimeric or humanized antibody of specificity at 4-1BBL.

3. pharmaceutical composition as claimed in claim 1 or 2, wherein this 4-1BBL has the sequence shown in SEQ ID NO:1 or 2.

4. pharmaceutical composition as claimed in claim 1, wherein this oligonucleotide has the SEQ of being selected from ID NO:10-15, the sequence of 22-38 and 45-56.

5. pharmaceutical composition as claimed in claim 1, wherein this solubility 4-1BB has amino acid/11 corresponding to (a) people 4-1BB polypeptide to amino acid/11 86; (b) amino acid 23 of people 4-1BB polypeptide is to amino acid/11 86; (c) amino acid/11 of mouse 4-1BB polypeptide is to amino acid/11 87; Or (d) amino acid 24 of mouse 4-1BB polypeptide to the sequence of amino acid/11 87.

6. pharmaceutical composition as claimed in claim 5, wherein this solubility 4-1BB has SEQ ID NO:7, the sequence shown in 8,20 or 21.

7. comprise carrier, the described 4-1BBL blocker of claim 1, and as the drug regimen of second medicament of anti-inflammatory drug.

8. drug regimen as claimed in claim 7, wherein this antiphlogiston is the antibody of specificity at tumour necrosis factor.

9. as claim 7 or 8 described drug regimens, wherein this antagonistic antibodies is the chimeric or humanized antibody of specificity at 4-1BBL.

10. as claim 7 or 8 described drug regimens, wherein this 4-1BBL has the sequence shown in SEQID NO:1 or 2.

11. as claim 7 or 8 described drug regimens, wherein this oligonucleotide has the SEQ of being selected from ID NO:10-15, the sequence of 22-38 and 45-56.

12. as claim 7 or 8 described drug regimens, wherein this solubility 4-1BB has amino acid/11 corresponding to (a) people 4-1BB polypeptide to amino acid/11 86; (b) amino acid 23 of people 4-1BB polypeptide is to amino acid/11 86; (c) amino acid/11 of mouse 4-1BB polypeptide is to amino acid/11 87; Or (d) amino acid 24 of mouse 4-1BB polypeptide to the sequence of amino acid/11 87.

13. drug regimen as claimed in claim 12, wherein this solubility 4-1BB has the sequence that is selected from SEQ ID NO:7-8 and 20-21.

14. specificity is at the chimeric or humanized antibody of 4-1BBL.

15. antibody as claimed in claim 14, wherein this antibody can be bonded to the 4-1BBL polypeptide that comprises SEQ IDNO:1 or 2.

16. have the SEQ of being selected from ID NO:10-15, the 4-1BBL blocker of the sequence of 22-38 and 45-56.

17. have amino acid/11 corresponding to (a) people 4-1BB polypeptide to amino acid/11 86; (b) amino acid 23 of people 4-1BB polypeptide is to amino acid/11 86; (c) amino acid/11 of mouse 4-1BB polypeptide is to amino acid/11 87; Or (d) amino acid 24 of mouse 4-1BB polypeptide to the solubility 4-1BB of the sequence of amino acid/11 87.

18. have SEQ ID NO:7, the described solubility 4-1BB of the claim 17 of 8,20 or 21 sequence.

19. reduce the method that inflammatory cytokine generates in the Mammals, it comprises the blocker to this administration 4-1BBL, wherein this 4-1BBL blocker is selected from the antagonistic antibodies of (a) specificity at 4-1BBL; (b) effective oligonucleotide that reduces the 4-1BBL expression of nucleic acid in cell; (c) solubility 4-1BB.

20. method as claimed in claim 19 further is included in and uses before this 4-1BBL blocker, identifies the Mammals that suffers from or easily suffer from diseases associated with inflammation.

21. as claim 19 or 20 described methods, wherein this inflammatory cytokine is tumour necrosis factor or IL-6.

22. as claim 19 or 20 described methods, wherein this Mammals is behaved.

23. as claim 19 or 20 described methods, wherein this antagonistic antibodies is the chimeric or humanized antibody of specificity at 4-1BBL.

24. as claim 19,20 or 23 described methods, wherein this 4-1BBL has the sequence of SEQ ID NO:1 or 2.

25. as claim 19 or 20 described methods, wherein this 4-1BBL blocker has the SEQ of being selected from ID NO:10-15, the sequence of 22-38 and 45-56.

26. as claim 19 or 20 described methods, wherein this solubility 4-1BB has amino acid/11 corresponding to (a) people 4-1BB polypeptide to amino acid/11 86; (b) amino acid 23 of people 4-1BB polypeptide is to amino acid/11 86; (c) amino acid/11 of mouse 4-1BB polypeptide is to amino acid/11 87; Or (d) amino acid 24 of mouse 4-1BB polypeptide to the sequence of amino acid/11 87.

27. method as claimed in claim 26, wherein this solubility 4-1BB has SEQ IDNO:7,8,20 or 21 sequence.

28. the method that inflammatory cytokine generates in the minimizing mammalian cell, it comprises this cell is contacted with the 4-1BBL blocker that wherein this 4-1BBL blocker is selected from the antagonistic antibodies of (a) specificity at 4-1BBL; (b) effective oligonucleotide that reduces the 4-1BBL expression of nucleic acid in cell; (c) solubility 4-1BB.

29. method as claimed in claim 28, wherein this inflammatory cytokine is tumour necrosis factor or IL-6.

30. as claim 28 or 29 described methods, wherein this Mammals is behaved.

31. as claim 28,29 or 30 described methods, wherein this antagonistic antibodies is the chimeric or humanized antibody of specificity at 4-1BBL.

32. as claim 28,29 or 30 described methods, wherein this 4-1BBL has the sequence of SEQ ID NO:1 or 2.

33. as claim 28 or 29 described methods, wherein this 4-1BBL blocker has the SEQ of being selected from ID NO:10-15, the sequence of 22-38 and 45-56.

34. as claim 28 or 29 described methods, wherein this solubility 4-1BB has amino acid/11 corresponding to (a) people 4-1BB polypeptide to amino acid/11 86; (b) amino acid 23 of people 4-1BB polypeptide is to amino acid/11 86; (c) amino acid/11 of mouse 4-1BB polypeptide is to amino acid/11 87; Or (d) amino acid 24 of mouse 4-1BB polypeptide to the sequence of amino acid/11 87.

35. method as claimed in claim 34, wherein this solubility 4-1BB has SEQ IDNO:7,8,20 or 21 sequence.

36. reduce the method for inflammation in the Mammals, it comprises the blocker to this administration 4-1BBL, wherein this 4-1BBL blocker is selected from the antagonistic antibodies of (a) specificity at 4-1BBL; (b) effective oligonucleotide that reduces the 4-1BBL expression of nucleic acid in cell; (c) solubility 4-1BB.

37. method as claimed in claim 36, wherein this Mammals is behaved.

38. method as claimed in claim 36, wherein this inflammation and rheumatoid arthritis, teenager's rheumatoid arthritis, psoriatic arthritis, psoriasis, ankylosing spondylitis, the Crohn disease, rheumatoid arthritis, systemic JCA, osteoporosis, or irritable bowel syndrome is relevant or caused by it.

39. method as claimed in claim 36, wherein this 4-1BBL has the sequence of SEQ ID NO:1 or 2.

40. method as claimed in claim 36, wherein this antagonistic antibodies is the chimeric or humanized antibody of specificity at 4-1BBL.

41. method as claimed in claim 36, wherein this 4-1BBL blocker has the SEQ of being selected from ID NO:10-15, the sequence of 22-38 and 45-56.

42. method as claimed in claim 36, wherein this solubility 4-1BB has amino acid/11 corresponding to (a) people 4-1BB polypeptide to amino acid/11 86; (b) amino acid 23 of people 4-1BB polypeptide is to amino acid/11 86; (c) amino acid/11 of mouse 4-1BB polypeptide is to amino acid/11 87; Or (d) amino acid 24 of mouse 4-1BB polypeptide to the sequence of amino acid/11 87.

43. method as claimed in claim 42, wherein this solubility 4-1BB has SEQ IDNO:7,8,20 or 21 sequence.

44. comprise the manufacturing article of container, comprise the 4-1BBL blocker in this container and this 4-1BBL blocker is being reduced the operation instruction of inflammatory cytokine in generating, wherein this 4-1BBL blocker is selected from the antagonistic antibodies of (a) specificity at 4-1BBL; (b) effective oligonucleotide that reduces the 4-1BBL expression of nucleic acid in cell; (c) solubility 4-1BB.

45. article as claimed in claim 44 wherein comprise explanation to this use of 4-1BBL blocker in inflammatory diseases to the explanation of the use of this 4-1BBL blocker.

46. article as claimed in claim 45, wherein this diseases associated with inflammation is a rheumatoid arthritis, teenager's rheumatoid arthritis, psoriatic arthritis, psoriasis, ankylosing spondylitis, the Crohn disease, rheumatoid arthritis, systemic JCA, osteoporosis, or irritable bowel syndrome.

47. as the described article of claim 44,45 or 46, wherein this inflammatory cytokine is tumour necrosis factor or IL-6.

48. as claim 44,45 or 46 described article, wherein this 4-1BBL has the sequence of SEQ ID NO:1 or 2.

49. as claim 44,45 or 46 described article, wherein this antagonistic antibodies is the chimeric or humanized antibody of specificity at 4-1BBL.

50. as the described article of claim 44,45 or 46, wherein this 4-1BBL blocker has the SEQ of being selected from ID NO:10-15, the sequence of 22-38 and 45-56.

51. as the described article of claim 44,45 or 46, wherein this solubility 4-1BB has amino acid/11 corresponding to (a) people 4-1BB polypeptide to amino acid/11 86; (b) amino acid 23 of people 4-1BB polypeptide is to amino acid/11 86; (c) amino acid/11 of mouse 4-1BB polypeptide is to amino acid/11 87; Or (d) amino acid 24 of mouse 4-1BB polypeptide to the sequence of amino acid/11 87.

52. as the described article of claim 44,45 or 46, wherein this solubility 4-1BB has SEQ ID NO:7,8,20 or 21 sequence.

53. the method for screening 4-1BBL blocker, it comprises:

(a) cell that will express 4-1BBL contacts with candidate's medicament; And

(b) detect the generation whether this candidate's medicament reduces inflammatory cytokine, perhaps whether this candidate's medicament prevents the 4-1BBL oligomerization, if wherein the generation of this candidate's medicament minimizing inflammatory cytokine maybe can prevent the 4-1BBL oligomerization, then this candidate's medicament is the 4-1BBL blocker.

54. method as claimed in claim 53, wherein this cell behaviour cell.

55. method as claimed in claim 53, wherein this cell is a scavenger cell.

56. method as claimed in claim 55, wherein this cell is the RAW264.7 cell.

57. the method for screening 4-1BBL blocker, it comprises:

(a) to administration candidate medicament; And

(b) detect the generation whether this candidate's medicament reduces inflammation or reduce inflammatory cytokine, if wherein this candidate's medicament reduces the generation of inflammation or minimizing inflammatory cytokine in this mammalian body, then this candidate's medicament is the 4-1BBL blocker.

58. method as claimed in claim 57, wherein this inflammation and rheumatoid arthritis, teenager's rheumatoid arthritis, psoriatic arthritis, psoriasis, ankylosing spondylitis, the Crohn disease, rheumatoid arthritis, systemic JCA, osteoporosis, or irritable bowel syndrome is relevant or caused by it.

59. as any described method of claim 53-58, wherein this inflammatory cytokine is tumour necrosis factor or IL-6.

60. as any described method of claim 53-58, wherein the generation of this inflammatory cytokine has reduced 20%, 25%, more than 30% or 30%.

61. as any described method of claim 53-58, wherein this 4-1BBL has the sequence of SEQ ID NO:1 or 2.

62. method as claimed in claim 57, wherein this Mammals is behaved.

Images