JPH10512142A - Anti-inflammatory CD14 polypeptide - Google Patents

Abstract

  (57) [Summary] The present invention relates to anti-inflammatory polypeptides, including soluble CD14-related polypeptides that have amino acids at positions 7-10 that differ from the native sequence or that have amino acids 1-14 deleted.

Description

DETAILED DESCRIPTION OF THE INVENTION                     Anti-inflammatory CD14 polypeptide Field of the invention   The present invention relates generally to the field of polypeptides having anti-inflammatory properties. CD1 Amino acids 7-10 of 4 may cause an inflammatory response in cells such as IL-6 production It has been found to contain important domains. The polypeptide of the present invention Based on replacing amino acids 7-10 of soluble form of CD14 with another amino acid . Other polypeptides of the invention lack the first 14 amino acids of soluble CD14. Have lost. The polypeptides of the invention are useful for treating inflammatory diseases such as sepsis. Can be used forBackground of the Invention   Sepsis is a life-threatening physical condition caused by infection or trauma. is there. Symptoms of sepsis include chills, heavy sweating, fever, weakness or reduced blood pressure. Yes, followed by leukopenia, intravascular coagulation, shock, adult dyspnea syndrome, It causes organ failure and often causes death. R. Ulevitch et al.,J. Trauma   30: S189-92 (1990).   Lipopolysaccharide ("LPS", "inner"), which is typically present in the outer membrane of all Gram-negative bacteria Toxins) are among the most studied and most understood sepsis-inducing substances I do. The exact chemical structures of LPS molecules obtained from various bacteria are species-specific and diverse. Regardless, a region called the lipid A region is common to all LPS molecules. E . Rietschel et al.Handbook of Endotoxins, 1 187-214, eds. R. Proctor and E.C. Rietsch el, Ed. Elsevier, Amsterdam (1984).   If not all of the LPS-dependent pathophysiological changes that characterize sepsis Many causes are in this lipid A region.   LPS is the primary cause of death in human patients with Gram-negative sepsis It is believed that. van Deventer et al.,Lancet, 1: 605 ( 1988); Ziegler et al.J. Infect. Dis., 136: 19- 28 (1987). Patients with sepsis and Gram-negative bacteremia for LPS Treatment with different monoclonal antibodies reduced mortality. Ziegler et al.,N . Eng. J. Med. , 324: 429 (1991).   Gram-positive bacteria also cause sepsis. Bone, R.A. C. Arch.In tern. Med. , 154: 26-34 (1994). Activation of the host cell Gram-positive cell walls or purified such as peptidoglycan and lipoteichoic acid Caused by extracellular cellular components. These substances are induced by LPS Induces a similar pattern of inflammatory response. Chin and Kosura,J. Immunol. 151: 5574-5585 (1993); Mattson et al. ,FEMS Immun. Med. Microbiol.7: 281-288 ( 1993); and Rotta,J. Z. Immunol. Forsch. Bd. , 149: 230-244 (1975). Cell wall material of LPS and Gram-positive bacteria Is a cytokine in polymorphonuclear leukocytes, endothelial cells and monocyte / macrophage cells. Such cell products are rapidly produced and released, and these products are Can initiate, regulate or mediate sexual and cellular immune responses and effects.   A specific support termed alpha-cachectin or tumor necrosis factor (TNF-α) Itokine is clearly the major mediator of septic shock. Beutler Et al.N. Eng. J. Med. , 316: 379 (1987). Inject LPS intravenously into laboratory animals and humans This causes a rapid, transient release of TNF-α. Beutler et al.J. Imm unol. , 135: 3972 (1985); Mathison et al.,J. Cli n. Invest. 81: 1925 (1988). Animals were challenged with anti-TNF-α antibody Pretreatment can modulate septic shock. Beutler et al.Scie nce , 229: 869 (1985); Mathison et al.,J. C1in. I nvest. 81: 1925 (1988).   Molecular receptors that can bind to sepsis-inducing substances and initiate some chemical reactions after binding The body plays a pivotal role in the pathogenesis of the symptoms of sepsis. CD14 Is strongly expressed on the surface of monocytes and macrophages and is the surface of granulocytes such as neutrophils. Is a weakly expressed 55-kD glycoprotein. S. M. Goyert et al.,J. Immunol. 137: 3909 (1986). A. Haziot et al.J . Immunol. 141: 547-552 (1988); M. Goyer t et al.Science  239: 497 (1988).   The cDNA and gene for human and mouse CD14 are cloned. And have been sequenced. E. FIG. Ferrero and S.M. M. Goyert ,Nuc. Acids Res.16: 4173 (1988); M. Goy ert et al.Science  239: 497 (1988); Setoguc hi et al.Biochem. Biophys. Acta  1008: 213-22 (1989). CD14 is a cleavable glycosylphosphatidylinositol Tail [A. Haziot et al.J. Immunol.141: 547-552 (1 988)], mature monocytes, macrophages, granulocytes and dendritic reticulocytes, It binds to the extracellular surface of the non-glomerular endothelium of the kidney, as well as the hepatocytes of the rejected liver.   CD14 mediates the response by binding to LPS. LPS and sCD1 4 shows a 1: 1 stoichiometry (Hailman, E. et al.,J. Exp . Med. 179: 269-277 (1994)). Production of TNF-α (Dentener, MA et al.,J. Immunol.7: 2885-2891 (1993)), production of IL-6 in astrocytes (Frey , E .; Et al.J. Exp. Med.176: 1665-1671 (1992)), Adhesion in endothelial cells Production of molecules (Frey, E. et al.,J. Exp. Med.176: 1665-16 71 (1992)) and of leukocyte integrins in PMNs (polymorphonuclear neutrophils). Activation (Hailman, E. et al.,J. Exp. Med.179: 269-27 7 (1994) is started. Spontaneous binding of LPS to CD14 is slow However, LBP can dramatically enhance this binding. LBP acts catalytically And one LBP transfers hundreds of LPS molecules to hundreds of CD14 molecules.   Another experiment isB. subtilis,S. aureus,as well asS. mitus Interaction of CD14 with gram-positive bacteria components such as (Pugin et al.,Immunity  1: 509-516 (1994) ). In addition, M. tuberculosis (Mycobacterium  tuberculos is The interaction between lipoarabinomannan derived from the cell wall and CD14 is also a cell activity Induces sexual development in a CD14-dependent manner (Zhang et al.,J. Clin. Invest . 91: 2076-2083 (1993); Pugin et al.,Immunity   1: 509-516 (1994)). These studies indicate that CD14 has a diverse bacterial structure. This suggests that the receptor recognizes the structure. CD Interaction of 14 with these structures initiates an inflammatory response in the host.   Some neutralizing monoclonal antibodies (mAbs) to CD14 are in v It is found to antagonize the cellular response to LPS in vitro and in vivo (For in vitro, Wright, SD et al.,Sciencec e   249, 1431-1433 (1990); Hailman, E .; Et al.J. Exp. Med. 179, 269-277 (1994); Frey, E .; A. La ,J. Exp. Med.176,1665-1671 (1992); Ardit i, M et al.Infect. Immun.61, 3149-3156 (1993) Wright, S .; D. Et al.J. Exp. Med.173,1281-128 6 (1991); Dentener, M .; A. Et al.J. Immunol  150 , 2885-2891 (1993); Grunwald, U.S.A. Et al.J. Immu nol Methods   155, 225-232 (1992), in v For Ivo, see Leturcq, D .; J. Et al.Satellite Mee ting of the 3rd Conference of the In ter national Endotoxin Society   22 (Abstra ct) (1994)); Wright et al.,Science  90: 1431-1 433 (1990)). Additional in vivo data includes CD14 neutralization mode. Animals injected with noclonal antibodies show reduced response to LPS and lack CD14 Lost mice proved not to respond to LPS. These experiments were performed on LP Release of inflammatory cytokines by inhibiting the interaction of S with membrane CD14 Suggests that it can be blocked.   In addition, CD14 is a soluble protein detected from normal serum or urine of nephrotic patients. It was also found to be present as a protein. Recent evidence indicates that sCD14 is LPS-dependent responses in cells lacking membrane CD14, such as vesicles and epithelial cells It has been shown that it can occur. When sCD14 binds to LPS in this type of cells, Promotes release of inflammatory cytokines and upregulation of adhesion molecules.   High concentration of sCD14 stimulates release of inflammatory cytokines from monocytes in whole blood assay A remarkable finding of blocking was obtained. SCD14 showed in this assay The beneficial effect is Attributed to its ability to keep LPS away from mCD14 on PMNs and PMNs Is done. Thus, sCD14, like the CD14 neutralizing monoclonal antibody, It could effectively block the interaction of LPS in CD14. However, s CD14 has another property of inducing inflammatory cytokines in endothelial cells And this property is a property of sCD14 for treating LPS-mediated inflammatory disorders. Restricts the use of. Thus, while maintaining the ability to bind LPS, If a sCD14 molecule is obtained that does not activate, this molecule will be useful in the treatment of inflammation. Must have outstanding properties.   Monoclonal antibodies complement identification of domains required for cell activation in sCD14 It can be an effective tool to help. We have found that mAbs MEM-18 and 3C SCD14 mutation 10 truncated at amino acid 152 And that the epitopes for these two mAbs were It was shown to exist within 152 amino acids (Juan, TS-C., Et al.). ,J. Biol. Chem.270, 1382-1387 (1995)). Departure The authors further note that the epitope for MEM-18 is between amino acids 57 and 64. The presence of this region and that this region is also essential for LPS binding (J uan, T .; S. -C. Et al.J. Biol. Chem.270, 5219-52 24 (1995)). Deletion of this region will result in LME as well as MEM-18 binding. PS binding has also been disrupted.   Epitope for mAb 3C10 Defines Another Functional Domain of CD14 You. This mAb is considered to recognize a region different from the recognition region of MEM-18. (Juan, TS-C. Et al.,J. Biol. Chem.270,138 2-187 (1995)). Monoclonal antibody 3C10 binds to sCD14 Does not affect LPS binding to sCD14 (Juan, TS et al. -C. Et al.J. Biol. Chem.270, 5219-5224 (1995) ). This suggests that this epitope may be involved in cell functions other than LPS binding Suggests.   For the reasons set forth above, one object of the present invention is to provide the symptoms of inflammatory diseases such as sepsis. And to develop effective treatment methods and therapies, including prevention of the disease. Also, sepsis Methods and therapies to effectively protect individuals who may suffer from such inflammatory symptoms Develop This is also one of the objects of the present invention.   Another object of the present invention is to provide LPS, Gram-negative bacteremia, Gram-positive bacterial cell components, Lamb positive bacteremia, mycobacterial lipoarabinomannan, mycobacteria Effectiveness including prevention of symptoms of infectious diseases and / or diseases mediated by CD14 To develop new treatment methods and therapies. Such diseases include ARD S, septic shock, acute pancreatitis, acute and chronic liver failure, intestinal or liver transplantation, There are inflammatory bowel disease, graft-versus-host disease of bone marrow transplantation and tuberculosis.Summary of the Invention   The inventors of the present invention are able to bind to lipopolysaccharide (LPS) 14 inhibits the binding of LPS or Gram-positive bacterial cell components to CD1 A group of polypeptides that reduce or eliminate the inflammatory response mediated by I found it. As used herein, the expression LPS binding inhibition refers to Gram-positive bacteria. Inhibition of binding to vesicle components is also meant. We have identified LPS binding domain in CD14. This group of polypeptides was designed based on the main important findings.   The polypeptide of the present invention preferably comprises a soluble form of CD14. Is a neutral amino acid, preferably hydrogen or C1-C6 Based on a neutral amino acid having an alkyl side chain of Polype of the present invention Peptides also include polypeptides starting at amino acid 15 in FIG. Figure 1 By reference, “soluble” CD14 (sCD14) is one of positions 1 to 6 Starts with one amino acid and ends with one of the amino acids from positions 152 to 348 Molecules selected from the following sequences: Native human CD14 is represented by X in FIG.₁-X_Four Glu-Leu-Asp-Asp. Preferably, the polypeptide Each of positions 7-10 of the amino acid is substituted with alanine.   The polypeptides of the present invention can bind to LPS, and consequently Can be prevented from binding to membrane CD14. Interaction between microbial cells and membrane CD14 When blocked, the cascade of events leading to inflammation, especially sepsis, is suppressed or prevented Is done. Therefore, the polypeptide of the present invention has anti-inflammatory properties   More particularly, the evidence presented herein is particularly relevant for amino acids 7- From polypeptide or soluble CD14 in which 10 regions have been replaced with alanine residues Polypeptides lacking amino acids 1-14 bind to LPS but respond to LPS Did Inflammatory response of cells mediated by the polypeptide, such as production of Itokine IL-6 The result shows that the result is substantially lower than that of native CD14.   In the examples below, we have identified a series of site-specific alani in sCD14. The 3C10 epitope is identified by creating a substitution mutant. Book We show that binding of 3C10 requires a region of amino acids 7-14. You. We further found that sCD14 suddenly has an alanine substitution at amino acids 7-10. Mutants (sCD14_{(7-10) A}) To characterize this domain by making did. This mutant was able to bind LPS, but was not able to bind to LPS. The ability to mediate vesicular responses was diminished.   The peptides and polypeptides of the present invention can be obtained by: (a) a standard synthesis method; (C) a recombinant method, (d) a combination of methods (a) to (c) or ( a) a combination of any one or more of (c) with another method for producing a polypeptide; Therefore, it can be manufactured.   The polypeptides of the present invention allow the polypeptide to be incorporated into a suitable pharmaceutical carrier substance. , Treatment like humans (or other mammals) Can be used for therapeutic or prophylactic purposes by administering an effective amount to patients requiring Can beBRIEF DESCRIPTION OF THE FIGURES   Accordingly, many other aspects and advantages of the present invention are set forth in the following detailed description with reference to the accompanying drawings. It will be clear from the description.   FIG. 1 is a sequence diagram of soluble human CD14 having 348 amino acids. Amino acid 7-10 (X₁-X_Four) Is Glu- in native mature human CD14. Leu-Asp-Asp. Amino acids 7-10 in the polypeptides of the invention are It may be any of the amino acids described herein.   FIG. 2 shows the sequence and expression of the sCD14 alanine substitution mutant.   FIG. 3 shows monoclonal antibody 3 against alanine substitution mutant of sCD14. FIG. 9 shows the results of BIAcore analysis of C10 binding. 4 days after electroporation Contained (MOCK), sCD14_1-348Or transfection with sCD14 mutant The recovered COS-7 cells were collected in conditioned medium (CM). For all commercials The ability to bind to antibody 3C10 was determined by the procedure described in the Examples section of this specification. Was analyzed. One experiment repeated four times The relative response unit (RRU) was recorded and calculated as the mean ± standard deviation.   FIG. 4 shows that mAb 3C10 was purified sCD14_{(7-10) A}Indicate that they do not recognize . A method for immobilizing mAb 3C10 on a sensor chip is described in the literature ( Juan, T .; S. -C. Et al.J. Biol. Chem.270,1382-1 387 (1995)). 10 μg / ml sCD14 for injection_1-348Or sC D14_{(7-10) A}It was used. Injection of solution at various 'stages' in sensorgram You. “Washing” indicates a washing step using HBS buffer as described in the Examples section. You. The experiment was repeated three times, showing the results of one experiment.   FIG. 5 shows that sCD14_{(7-10) A}Lack the ability to generate a cellular response to LPS Indicates that it is damaged. A. sCD14_{(7-10) A}Then, IL by U373 cells The ability to stimulate -6 production is reduced. U373 cells were transformed with LPS (20 ng / m Various concentrations of sCD14 in the presence or absence of l)_1-348Or sCD14_{( 7-10) A} For 24 hours. Measure IL-6 levels according to procedures described in the literature (Juan, TS-C. Et al.,J. Biol. Chem.270,138 2-13 87 (1995)). The data presented was obtained by repeating one experiment three times. The mean ± standard deviation of the four readings obtained. B. sCD14_1-348Is L Mediates PMN responses to PS and LBP, but not to sCD14_{(7-10) A}Is an intermediary do not do. Newly isolated PMNs are converted to "smooth" LPS (E. coli) 0111: B4 30 ng / ml), rLBP (1 μg / ml) and designated concentration Degree sCD14_1-348Or sCD14_{(7-10) A}For 10 minutes at 37 ° C I was vate. Wash cells and measure adhesion to fibrinogen-coated wells (Hailman, E. et al., J. Exp. Med. 179, 269-277). (1994), 25). Error lines indicate the standard deviation of three overlapping measurements.   FIG. 6 shows that sCD14_{(7-10) A}Does not activate NF-κB. Various Treatment (lane 1, control; lane 2, LPS; lane 3, sCD14_1-348; Leh 4, sCD14_1-348And LPS; Lane 5, sCD14_{(7-10) A}Lane 6, s CD14_{(7-10) A}And LPS; Lane 7, sCD14_{Δ (57-64) A}Lane 8, sC D14_{Δ (57-64) A}And LPS) to collect whole cell extracts of U373 cells Marking by the procedure described in the Examples section The protein was bound to the NF-κB oligonucleotide. 4.5% native po The complex of NF-κB was separated on a acrylamide gel. After electrophoresis, dry the gel Dry and expose to X-ray film for 16 hours. Complex of labeled probe and NF-κB Is shown.   FIG. 7 shows that sCD14_{(7-10) A}But^ThreeShows that it forms a stable complex with H-LPS You. Using the procedures described in the Examples section, various concentrations of sCD14_1-348(Lane 2 -4) or sCD14_{(7-10) A}(Lane 5-7) at 3 μg / ml^ThreeH-LPS Together with the ink in the absence (A) or presence (B) of 16.7 nM rLBP Was added. Lane 1 contains LPS in the absence of additional protein. The mixture Run on 4-20% native polyacrylamide gel for fluorography Processed. Location of LPS not forming a complex and LPS and sCD14_1-348Ma Or sCD14_{(7-10) A}Indicates the position of the complex with   FIG. 8 shows that sCD14_{(7-10) A}4 shows the inhibition of LPS-induced cell response by. A. s CD14_{(7-10) A}Inhibits LPS-induced PMN adhesion. Rough LP S (Salmonella  minnesota  R60, 10 ng / ml) , LBP And various concentrations of sCD14_1-348Or sCD14_{(7-10) A}With PMN Incubated at 37 ° C. for 30 minutes before adding. Follow the procedure described in the Examples section. The adhesion of PMN to ibrinogen was measured. Error bars are three readings The standard deviation is shown. B. sCD14_{(7-10) A}Of TNF-α production in whole blood by Inhibition. Following the procedure described in the Examples section, 250 μl of whole blood was Albumin, sCD14_1-348Or sCD14_{(7-10) A}Together with 0.25 ng / m l smooth LPS (Salmonella  minnesotaWild type) After incubation at 37 ° C. for 3 hours, TNF-α production was measured. TNF- α production ratio is determined by adding TNF-α produced in the presence of It is shown as a ratio divided by TNF-α produced in the absence of protein. error The line is the standard deviation generated at the six readings.   FIG. 9 shows that Gram-positive bacterial cell components compete with LPS for binding to sCD14. Indicates that they match.^ThreeSCD14 (50 μl) containing H-LPS (1 μg / ml) g / ml) alone (lane 1) or with LPS (lane 2) orSt aphylococcus   aureusWith the crude extract (SACE) (Lane 3-6) Incubation at 37 ° C. for 17 hours in PBS supplemented with 1 mM EDTA. Cubbed. The sample is then run on a native polyacrylamide gel, The position of the radioactive band was determined by radioautography.Detailed Description of the Preferred Embodiment   The present invention relates to the cell components of Gram-positive bacteria or Gram-negative bacteria (eg, Gram-negative bacteria). Mediated by CD14, such as IL-6 production in response to CD14 region involved in inflammatory cell response discovered by the present inventors based on. The evidence supporting these findings is described in detail in the examples below.   FIG. 1 contains the LPS binding and IL-6 induction regions corresponding to amino acids 7-10 FIG. 2 shows a sequence diagram of human CD14.   The polypeptides of the present invention may be located at positions 7-10 (inclusive) of soluble CD14. Substitute or replace amino acids with amino acids different from those in the native molecule Based on deleting the first 14 amino acids of soluble CD14. See FIG. Then, amino acids 7-10 in the polypeptide containing the substituted amino acid are Glu- Not Leu-Asp-Asp. Preferably, the substitution The amino acids are neutral amino acids. The side chains of these neutral amino acids are preferably water Selected from the group consisting of nitrogen and alkyl having 1 to 6 carbon atoms, The alkyl group may be a halogen (e.g., Cl, Br, I), -OH, -CN, -OR (R = Alkyl having 1 to 6 carbon atoms) and related structures (tures) may be substituted with one or more substituents selected from The resulting polypeptide is capable of binding LPS and / or flame in monocytes and PMNs Inhibits the release of disease mediators and simultaneously triggers inflammatory responses in endothelial and epithelial cells Amino acids 7-10 as long as they show a reduced ability to perform (compared to wild-type sCD14) The details of the amino acid to be substituted are not critical. First one of soluble CD14 The polypeptide of the present invention, in which four amino acids have been deleted, corresponds to the position 152 in sCD14. 348 to one amino acid. Preferred deletion peptides are 1, amino acids 15-152 and 15-348 of FIG.   The remainder of the polypeptide (other than the substituted amino acids) generally comprises the native soluble CD Equals 14. The term "soluble CD14 (sCD14)" refers to positions 1-6 in FIG. From 152 to 348 Means the polypeptide corresponding to the amino acid in. In this document, It was noted that the amino acid numbers used therein correspond to the amino acid sequence of FIG. No. For example, if a particular soluble CD14 starts at amino acids 2-6 shown in FIG. If any, the amino acids to be replaced are amino acids 7-10 of the complete sequence of FIG. Is the amino acid corresponding to   For the purposes of the present invention, the molecules shown in FIG._1-348 Call. Other specific examples of soluble CD14 include starting amino acids based on the numbering diagram of FIG. Named by the terminating amino acid, for example, sCD14_1-152SCD14;_2-152; sCD14_15-152SCD14;_15-348And so on. The amino acids in FIG. 1 have been substituted The polypeptides are named as follows. For example, amino acids 7-10 in FIG. SCD14 substituted by Nin_1-348Is sCD14_{1-348 (7-10) A}It is named. this" A "is a one-letter code for the amino acid alanine. Position 7 and 9-10 SCD14 with alanine at position 8 and glycine at position 8_1-348Is sCD14_{1-348 (7,9,10) A (8) G} It is named.   Some preferred specific examples of the polypeptide of the present invention are shown below. sCD14_{1-348 (7-10) G}                sCD14_{1-152 (7-10) G} sCD14_{1-348 (7-10) A}                sCD14_{1-152 (7-10) A} sCD14_{1-348 (7-10) V}                sCD14_{1-152 (7-10) V} sCD14_{1-348 (7-10) L}                sCD14_{1-152 (7-10) L} sCD14_{1-348 (7-10) I}                sCD14_{1-152 (7-10) I} sCD14_{1-348 (7-10) P}                sCD14_{1-152 (7-10) P} sCD14_15-348                      sCD14_15-152   The present invention also relates to the physiologically acceptable polypeptides disclosed herein. Salt. Also, one or more D or L A mino acid may be included. However, all amino acids have the L configuration Is preferred.   The polypeptides of the present invention have an intracellular inflammatory response compared to native sCD14. Is expected to have the ability to reduce To conveniently measure reduction in inflammation For example, using the method described in Example 4 below, IL- 6 is measured. IL-6 is preferably 1 compared to native sCD14. It will be reduced to / 5 or less, particularly preferably to 1/10 or less.   Also, in each case, the ability to induce IL-6 (or related cytokines) is low. Amino acids are chemically induced as long as the underlying LPS binding is maintained It may be embodied. Thus, the term "chemical derivative" of a polypeptide of the invention Is included within the scope of the term "polypeptide" as used herein. This These chemical derivatives are represented by X₁-X_FourAmino acid is a substituted polypeptide part Contains additional chemical moieties.   Covalent modifications of the polypeptide are included within the scope of the invention. Such a modification Reacts a target amino acid residue of the polypeptide with a selected side chain or terminal residue. It can be introduced into the molecule by reacting with the obtained organic derivatizing agent.   The histidyl residue is reacted with diethyl pyrocarbonate at pH 5.5-7.0. Derivatization. The reason is that this substance is compared to the histidyl side chain. This is because it is specific. Parabromophenacyl bromide is also useful. Preferably Initiates the reaction in 0.1 M sodium cacodylate at pH 6.0.   Lysinyl and amino terminal residues are incompatible with succinic or other carboxylic anhydrides. Respond. Derivatization with these substances has the effect of reversing the charge of ricinyl residues. Have. Alpha-amino -Other suitable reagents for derivatizing the containing residues include methylpicolinimidate Imidoesters such as pyridoxal phosphate, pyridoxal, chlorobo Rohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4 pen Reaction with glyoxylate catalyzed by transaminases with tandione There is.   Arginyl residues are modified by reaction with one or more conventional reagents . Examples of reagents include phenylglyoxal, 2,3-butanedione, 1,2 -Cyclohexanedione and ninhydrin. Derivatization of arginine residues In this case, the pK of the guanidine functional group is high, so it is necessary to react under alkaline conditions. You. In addition, these reagents include the group of lysine and the epsilon-amino group of arginine. Can react with   In particular, the modification of tyrosyl residues themselves has been studied eagerly, and Tyrosyl residues by reaction with compounds or tetranitromethane Is of particular interest. O-acetyltyrosyl species and 3-d To form the toro derivatives respectively, N-acetylimidazole and tetranitro Methane is frequently used.   The carboxyl side group (aspartyl or glutamyl) is 1-cyclohexyl -3- (2-morpholinyl- (4-ethyl) carbodiimide or 1-ethyl -3- (4-azonia-4,4-dimethylpentyl) carbodiimide Selectively modified by reaction with rubodiimide (R'-N-C-N-R ') . In addition, aspartyl and glutamyl residues can react with ammonium ions. And is converted to asparaginyl and glutaminyl residues.   Glutaminyl and asparaginyl residues are often deamidated to the corresponding groups. It becomes rutamyl and aspartyl residues. Alternatively, these residues can be It may be deamidated. Either form of these residues is included within the scope of the present invention. It is.   Derivatization with bivalent reagents makes peptides or their functional derivatives water-insoluble It is useful for cross-linking to a porous support matrix or other polymeric carrier. Regular use Examples of the crosslinking agent include 1,1-bis (diazoacetyl) -2-phenylethane , Glutaraldehyde, N-hydroxysuccinimide ester such as 4- Ester with azidosalicylic acid, 3,3'-dithiobis (succinimidyl propyl Disuccinimidyl such as onate) Ester-containing homobifunctional imide ester, bis-N-maleimido-1,8-o There are divalent maleimides such as butane. Methyl-3-[(p-azidophenyl Derivatives such as) (dithio) propioimidate form crosslinked structures in the presence of light. This produces a photoactivatable intermediate that can be formed. Or charcoal activated with cyanogen bromide Reactive water-insoluble matrices such as hydrates and US Pat. No. 3,969,28 No. 7, No. 3,691,016, No. 4,195,128, No. 4,247,64 No. 2, No. 4,229,537 and No. 4,330,440. The reactive substrate used is used for protein immobilization.   Other modifications include hydroxylation of proline and lysine, seryl or Phosphorylation of hydroxyl group of threonyl residue, oxidation of sulfur atom of Cys, Methylation of the alpha-amino group of the glycine, arginine and histidine side chains (T.E. Creighton,Proteins: Structure and  Mol ecule Properties , W.S. H. Freeman & Co. , S an Francisco, p. 79-86 (1983)), of the N-terminal amine Acetylation and, in some cases, amidation of the C-terminal carboxyl group. You.   The activity of the polypeptide variant is determined by appropriate screening assays based on the desired properties. Screen for gender. Produced in a suitable bioassay as described herein Screen for physical activity. For example, LPS and CD14 (or the present invention) Polypeptide) may be measured in a standard competitive binding assay. cell The activity of reducing the inflammatory response of cells (e.g., U3 73 cells).   Redox or thermal stability, hydrophobicity, sensitivity to proteolysis, Are likely to aggregate with the carrier or as a multimer. Changes in repeptide properties are assayed by methods well known to those of ordinary skill in the art.   Such derivatized moieties improve solubility, absorption, biological half-life, etc. I can do it. Or, such parts may eliminate unwanted side effects of the protein. Or it can be reduced. Portions that can mediate such effects include, for example, Remingto n, Pharmaceutical Sciences, 16th. , M ack Publishing Co. , Easton, Pa (1980) It is shown.   The polypeptides of the present invention may also include polypeptides, non-CD proteins, linear polypeptides. (Polyethylene glycol, polylysine, etc.), branched polymers (eg, U.S. Pat. No. 4,2,2, issued Sep. 15, 1981 to Denkenwalter et al. No. 89,872, issued to Tam on U.S. Pat. No. 490, published Oct. 28, 1993, by Frechet et al., WO 93/212. 59), lipids, cholesterols (eg steroids), or carbohydrates Alternatively, it can be covalently or non-covalently linked to a carrier molecule such as an oligosaccharide.   Polypeptides of the invention are expected to have the ability to bind LPS . This binding binds LPS to macrophage membrane CD14 (mCD14) And an anti-inflammatory response occurs in the mammal. Furthermore, In the polypeptides of the present invention, mCD14 deficiencies such as endothelial cells and epithelial cells The ability to trigger an inflammatory response during cell loss is reduced. Polypeptide of the invention Is also expected to bind to the cellular components of Gram-positive bacterial cells that cause inflammation. (These cellular components are related to LPS, but are The structure (s) giving rise to the inflammatory response is unclear is there). The term "binding" to LPS is a standard competitive assay, The peptide has an L between 1 mM and 1 nM, preferably between 100 μM and 10 nM. Ability to inhibit binding of CD14 to PS by 50% (IC₅₀Value). Standard binding assays are performed according to procedures well known in the art.   The polypeptide of the present invention can be produced by various methods. For example, using solid-phase synthesis technology Can be used. Suitable techniques are well known in the art and are described, for example, in Merrifield,Chem. Polypeptides, pp. 335-61 (Katsuoyan Nis and Panayotis ed. 1973): Merrifield,J. A m. Chem. Soc. , 85, 2149 (1963); Davis et al.,Bio chem. Int'l , 10, 394-414 (1985); Stewart. And Young,Solid Phase Peptide Synthesi s (1969); U.S. Pat. No. 3,941,763; Finn et al.,The Pr oteins , 3rd ed. , Vol. 2, pp. 105-253 (1976) And Erickson et al., The Proteins, 3rd ed. , V ol. 2, pp. 257-527 (1976).   In addition, polypeptides can be produced in transformed host cells using recombinant DNA technology. It is highly preferred. To this end, a recombinant DNA encoding the polypeptide is used. Create a molecule. Methods for making such DNA molecules are well known in the art. example For example, a sequence encoding a polypeptide can be excised from DNA with an appropriate restriction enzyme. Can be Alternatively, using chemical synthesis techniques such as the phosphoramidite method DNA molecules can be synthesized. Also, these techniques can be used in combination.   The invention also includes a vector capable of expressing the peptide in a suitable host. Be Is a peptide encoding a peptide operably linked to an appropriate expression control sequence. Consists of NA molecules. This action occurs before or after inserting the DNA molecule into the vector. Methods for effecting the binding of possible regulatory sequences are well known. Promoted expression control sequences Promoter, activator, enhancer, operator, ribosome binding site, A start signal, an end signal, a cap signal, a polyadenylation signal, and There are other signals involved in the regulation of transcription or translation.   An appropriate host is transformed using the obtained vector carrying a DNA molecule. This Transformation of DNA is performed by a method well known in the art. No.   Any of a number of known host cells available can be used to practice the present invention. You may. The choice of a particular host depends on a number of factors recognized by the art in the art. It depends on the factor. Such factors include, for example, compatibility with the selected expression vector. Compatibility, toxicity of the peptide encoded by the DNA molecule to the vector, Has transformation speed, ease of peptide recovery, expression characteristics, biosafety and cost . Not all hosts are equally effective in expressing a particular DNA sequence. It is necessary to understand these factors and balance these factors.   Useful microbial hosts that meet these general guidelines include bacteria (eg, E. coli species). , Yeast (for example,SaccharomycesSpecies), and other fungi, insects, plants, There are cultured cells of mammals (including humans) or other hosts known in the art.   Next, the transformed host is cultured under conventional fermentation conditions so that the desired peptide is expressed. Nourish. Such fermentation conditions are well-known in the art.   Finally, the polypeptide is purified from the culture. These purification methods are well known in the industry. Is knowledge.   The polypeptide of the present invention is required to bind to LPS / gram-positive bacterial cell components. It can be used in any of a number of situations. For example, inflammatory bowel disease, sudden Sexual and chronic liver failure, graft versus host disease (bone marrow transplant), intestinal or liver transplant, AR Therapeutic and prophylactic use of polypeptides against DS, acute pancreatitis and tuberculosis . A particularly preferred target disease is septic shock.   The novel polypeptides are useful for preventing or treating septic shock in mammals, including humans. For treatment, a dose of about 0.1 to 100 mg / kg body weight, preferably about 1 to 5 It is effective at the level of 0 mg / kg and this amount is divided, for example, on a daily dose basis. It may be administered in doses. The polypeptide is produced in a manner that causes septic shock. May be administered prophylactically to patients who may or may be in contact with an object Or to detoxify LPS (bacterial endotoxin) by using the same dose as above In addition, it is desired to detoxify or prevent endotoxin contamination in vivo or in vitro. It may be done at a level effective to achieve the result. The amount is about 1 mole of poisoning Based on routine experimental procedures, based on the assumption that one mole of polypeptide binds to Can be determined based on the A particular dose of a particular polypeptide will depend on the particular use or disease Weight Within the range specified herein or outside the range depending on the severity and physical condition of the host, etc. Can change. One skilled in the art will be able to ascertain the proper dose using standard procedures.   A pharmaceutical composition of the present invention can be administered by any means that serves its intended purpose. For example, subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intracapsular, transdermal, or buccal And the like. Alternatively or simultaneously, by oral or rectal route You may. Pharmaceutical compositions may be used for bolus injection or long-term gradual perfusion. Thus, they may be administered parenterally.   These pharmaceutical compositions comprise, in addition to the polypeptide, a suitable pharmaceutically acceptable carrier. The carrier can be used to easily process the active ingredient into a pharmaceutical preparation that can be used as a medicament. Consists of auxiliaries and excipients. Formulations, especially tablets, sugar-coated tablets, capsules Formulations that can be administered orally and can be used in preferred dosage forms, such as rectal administration such as suppositories Formulations and suitable solutions that can be administered by injection or orally are suitable. Preferably about 0.1 to about 99%, more preferably about 25 to 85% (one or more) The active compound is contained with the excipient.   Suitable excipients include, in particular, sugars such as lactose, sucrose Fillers such as glucose, mannitol or sorbitol; cellulose preparations; And / or phosphoric acid such as tricalcium phosphate or calcium hydrogen phosphate Lucium; corn starch, wheat starch, rice starch, potato starch, gelatin , Tragacanth gum, methylcellulose, hydroxypropyl methylcellulose , Sodium carboxymethylcellulose, and / or polyvinyl pyrrolide There are binders, such as starch pastes, prepared with the aid of starch. If desired, the residue Powder, carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, or algi Disintegrants may be added, such as acids or salts such as sodium alginate. Auxiliaries which can be used in the composition according to the invention include silica, talc, stearic acid. Lubricants and flow modifiers, such as, for example, There are surfactants such as polyethylene glycol.   Based on the teachings contained herein, the teachings of the present invention may be tailored to a particular task or situation. It will be appreciated that the use is within the capabilities of those skilled in the art. Fruit of the substance of the present invention Examples will be described below.                                  Example                               Materials and methods reagent   Recombinant soluble CD14 (rsCD14) and recombinant LBP (rLBP) ilman, E. et al., J. Exp. Med. 179, 269-277 (1994). . Concentrations of all purified proteins were determined using Micro BCA Pro as described by the manufacturer. It was measured with a tein kit (Pierce, Rockford, IL). Full length rsCD14 Ends at position 348 of the mature protein (Hailman, E. et al., J. Exp. Med. 179, 269- 277 (1994))._1-348That. Anti-CD14 mAb 3C 10 from the American Type Culture Collection (ATCC TIB 228) Purified from cell line conditioned medium by chromatography on protein G Was. Rabbit polyclonal anti-human CD14 antiserum was obtained from Juan, T.S.-C. et al., J. Biol. hem. 270, 1382-1387 (1995). Rough LPS (Salmonella min nesota  R60 or Re595) and smooth LPS (E. coli 0111: B4 orSalmonellamin nesota Wild type) is LIST Biological Laboratories (Campbell, CA). The enzyme for DNA manipulation is Boehringer Mannheim (In dianapolis, IN).Site-directed mutagenesis   Nine alanine substitution mutants of sCD14 were used in this study. Figure 2 shows each mutation Summarize body name and substituted amino acid residues. Transformer site-specific Mutagenesis kits (Clontech, Palo Alto, CA) were previously described (Juan, T.S.-C Et al., J. Biol. Chem. 270, 5219-5224 (1995)). Coding for an alanine substitution mutant of sCD14 cloned in NA was prepared. The primers used for each mutant are as follows: Transient expression of sCD14 mutant protein in COS-7 cells   In order to express the sCD14 mutant protein, the sCD14 mutant cDNA was Containing mammalian expression vector into COS-7 (ATCC CRL 1651) cells. Introduced by electroporation. Electroporation conditions and transformer Generation of serum-free CM from transfected COS-7 cells is described in Juan, T.S. .-C. Et al., J. Biol. Chem. 270,1382-1387 (1995). Mutant sCD 14 was analyzed by Western blot using an anti-CD14 polyclonal antibody. Was analyzed.BIAcore analysis of the interaction of 3C10 mAb with sCD14 mutant   Recognition of sCD14 mutant protein by neutralizing monoclonal antibody 3C10 , BIAcore biosensor equipment Experiments Device, CM5 sensor chip and amine binding kit It was purchased from cia Biosensor (Piscataway, NJ). In summary, mAb 3C10 (200 μg / mL in 20 mM sodium acetate, pH 3.4) was added by the manufacturer. Immobilized on a CM5 sensor chip by amine binding as described. Or that Then, the flow cell in which 3C10 was immobilized was sequentially added with the detailed solution in the following steps. Incubated: Step 1, COS-7 CM, 2 minutes and Step 2, H BS buffer [10 mM N-2-hydroxyethylpiperazine-N'-2-eta Sulfonic acid, pH 7.5, 0.15 M NaCl, 3.4 mM EDTA, 0 . 005% (v / v) surfactant P20 (Pharmacia Biosensor)] for 2 minutes. For regeneration, a 10 mM HCl solution was injected for 2 minutes. Injection is 5 μL / min Performed at a flow rate. SCD14 mutant in COS-7 CM to immobilized 3C10 Relative response units (RRU) were calculated to quantify binding. CM injection And immediately before CM injection from the response unit (RU) recorded after washing for 2 minutes The obtained RU was subtracted to obtain an RRU.Purification of sCD14 _{(7-10) A}   sCD14_{(7-10) A}An expression vector containing a cDNA encoding J. Exp. Med. 179, 269-277 (1994). Stably transfected into Chinese hamster nest (CHO) cells. Serum-free culture A single clone was grown in the ground and sCD14_{(7-10) A}Produced CM containing . Anti-CD14 polysaccharide bound to Sepharose 4B (Pharmacia, Piscataway, NJ) Except for immunoaffinity chromatography using lonal antibodies And exactly Juan, T.S.-C. Et al., J. Biol. Chem. 270, 5219-5224 (1995) Next, the mutant protein was purified. The purity of the sample was determined by SDS-polyacrylamide. Dogel electrophoresis (SDS-PAGE) followed by silver or Coomassie blue staining Checked. The altered amino acid sequence was verified by N-terminal sequencing.U373 bioassay   Growth of U373 cells (ATCC HTB17, Rockville, MD), purified sCD1 Activation of the four preparations and quantification of IL-6 was performed as described in Juan, T.S.-C., et al., J. Biol. Chem. . 270, 1382-1387 (1995). Summary If sCD14_1-348Or sCD14_{(7-10) A}And LPS mixture in medium without serum Added to subsurface U373 cell monolayer and incubated for 24 hours. Then the supernatant Of IL-6 was measured by ELISA.Polymorphonuclear leukocyte (PMN) adhesion assay   RLBP that can adhere PMN to a plate coated with fibrinogen sCD14_{(7-10) A}Or sCD14_1-348The ability of a previously established protocol (Hailman, E. et al., J. Exp. Med. 179, 269-277 (1994); Juan, T.S.-C., et al., J. Biol. Chem. 270, 1382-1387 (1995)). In summary, PMN is replaced by LP S, rLBP, and sCD14_{(7-10) A}Or sCD14_1-348And incubate for 10 minutes Hailman, E. Bait, wash and adhere to fibrinogen coated surfaces. . J. Exp. Med. 179, 269-277 (1994); Juan, T.S.-C. Et al., J. Biol. Chem. 270, It was measured as described in 1382-1387 (1995). This protocol uses smooth LPS. When used, the adhesion is completely sCD14_1-348(Juan, T.S.-C. et al., J. Bi ol. Chem. 270, 1382-1387 (1995)).   SCD14 at high concentrations binds to LPS and inhibits LPS-mediated PMN adhesion_{(7-10) A} Or sCD14_1-348Was also evaluated. In this experiment, before adding PMN, Rough LPS (Salmonella minnesota R60, 10 ng / ml) with rLBP (1 μg) / Ml) of sCD14_{(7-10) A}Or sCD14_1-34830 at 37 ° C Incubated for minutes. PMN adhesion was measured as described above.Electrophoretic mobility shift assay   A whole cell extract from U373 cells was prepared and the activity of the transcription factor NF-κB was determined. Sexualization was evaluated. One day before stimulation, in a 6-well plate at a density of 1 million cells / well Cells were inoculated. For stimulation, purified sCD14_1-348, SCD14_{Δ (57-64)}( Juan, T.S.-C. et al., J. Biol. Chem. 270, 5219-5224 (1995)), or sCD14_{(7-10 ) A} At a final concentration of 20 ng / ml in the presence of 20 ng / ml of Re595 LPS. Or added over 20 hours in the absence. Cells in 1 × PBS (GIBCO-BRL) Wash twice, lysis buffer supplemented with 1% Triton X-100 (Sigma) (20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 1 mM  EDTA, 0.5 mM dithiotrate 1 mM Pefabloc (Boehrinqer Mannheim), 5 μg / mL Leupe Putin, 1 mM sodium orthovanadate, 2 μg / mL aprotinin) 2 Scraped in 00 μL. Transfer the crude extract to a microcentrifuge tube and remove cell debris. Separation was performed by centrifugation at 14,000 × g and 4 ° C. for 10 minutes. Quickly extract liquid And stored at -80 ° C. Check the protein concentration of the whole cell extract. It was 1.5-2 μg / μL as measured by the black BCA assay.   To test the NF-κB complex, an electrophoretic mobility shift assay was performed . Two oligonucleotides: Anneal the human immunodeficiency virus long terminal repeat promoter ( NF-κB binding site of Nabel, G. and Baltimore, D. Nature 326, 711-713 (1987)). Was produced. Then, this annealed DNA flag The Klenow fragment (Boehrinqer Mannheim) and α-³²PdCTP (Amersham, Arlinqton Heiqhts, IL) This was used as a probe at a concentration of 50,000 cpm / lane (about 25 fmole). used. For binding, 4 μL of whole cell extract was added to 5 × binding buffer (1 50 mM Tris-HCl, pH 8.0, 40 mM MgCl_Two, 5 mM DTT 4%, (poly-dI-dC): (poly-dI-dC) (ph armacia, piscataway, NJ) 2.5 μg, radiolabeled DNA probe, and lysis buffer The buffer was incubated with an appropriate volume (final volume was 20 μL / reaction). Reaction liquid Was incubated in a 30 ° C. water bath for 30 minutes, and the complex was allowed to reach 0.5 × TBE (50 m M Tris-HCl, pH 8.0, 45 mM boric acid, 5 mM EDTA) Separation was performed on a native 4.5% polyacrylamide gel (30 mA, 2 hours). Thereafter, the gel was vacuum dried at 80 ° C. for 1 hour and exposed to Kodak X-ray film for 20 hours. Glowed. . In competition experiments, a 100-fold molar excess of unlabeled prior to addition of the radioactive probe The NF-κB probe was pre-incubated for 10 minutes.Native PAGE assay   To directly assess LPS binding of purified sCD14 preparations, sCD14_1-348 Or sCD14_{(7-10) A}The various At the concentrations (0, 101, 303, 909 nM)E.coliFrom K12 stock LCD25 Prepared^ThreeH-LPS (List Biological Laboratories) 3 μg / mL, 16 . Incubated in the presence or absence of 7 nM rLBP. 37 At 30 ° C. for 30 minutes, then undenatured 4-20% polyacrylamide The gel was electrophoresed on a gel. The gel was previously described in Hailman, E. et al., J. Exp. 179,269-27 Prepared for fluorography as described in 7 (1994).Inhibition of LPS-induced TNF-α production in whole blood   The ability of sCD14 to bind to LPS and inhibit TNF-α production in whole blood Are described in Haziot, A. et al., J. Immunol 152, 5868-5876 (1994). Simple Briefly, various concentrations of 50 μL RPMI medium (GIBCO-BRL, Gaithersb. urg, MD), bovine serum albumin (Miles, New Haven, CT), sC D14_1-348Or sCD14_{(7-10) A}Using heparin as an anticoagulant, Added to 50 μL of freshly collected blood. Smooth LPS (Salmonella minnesotafield Neat) was added at a final concentration of 0.25 ng / mL. The reaction was incubated at 37 ° C for 3 hours. Niki And centrifuged at 16,000 × g for 2 minutes to obtain a supernatant. TNF- of supernatant The α concentration was determined by the Quantikine TNF-α ELISA kit as specified by the manufacturer. (R & D Systems, Minneapolis, MN).                               Example 1 The alanine substitution of amino acids 7-10 or 11-14 can be achieved by neutralizing mAb 3C10 and C Completely prevents binding to D14   3C10 is a mAb that recognizes the N-terminal 152 amino acids of CD14 (Juan , T.S.-C. et al., J. Biol. Chem. 270, 1382-1387 (1995)). 3C10 is sCD14_{1- 348} Previous experiments have shown that it neutralizes the activity of S.D. (Wright, S.D. et al., Science 249). , 1431-1433 (1990); Hailman, E. et al., J. Exp. Med. 179, 269-277 (1994); Frey, E.A. Et al., J. Exp. Med. 176, 1665-1671 (1992); Wright, S.D., et al., J. Exp. Med. 173,128 1-1286 (1991)). Neutralization of sCD14 activity is an epitope within the N-terminal 152 amino acids To demonstrate that this is due to the binding of_1-348Or sCD 14_1-152Inhibiting U373 cell IL-6 production mediated by We have demonstrated (data not shown).   To determine the epitope for mAb3C10, a series of alanine substitution mutations Bodies were generated by site-directed mutagenesis (FIG. 2). Coding various sCD14 mutants Transfecting COS-7 cells with a plasmid containing the cDNA sequence to be CM from these cells was analyzed for expression of sCD14 mutein by Western blotting. Tested by blotting. sCD14_{(18-21) A}All sCD14 variants except It was expressed and secreted in COS-7 cells (FIG. 2). Then, BIAcore analysis (FIG. 3) ) Was tested for the ability of CM containing mutant sCD14 to bind to 3C10. sCD14_{(7-10) A}Or sCD14_{(11-14) A}Does not bind to 3C10 It was found that These data indicate that the region between amino acids 7-14 is 3C10 Suggests that it is involved in recognizing.                               Example 2 Purification and characterization of sCD14 _{(7-10) A}   Since neutralizing mAb 3C10 recognizes amino acids 7-14, we believe that C That this region of D14 may play an important role in the biological activity of CD14 Was concluded. To help understand the role of this region, sCD14₍₇ -10) A stable CHO cell line expressing A was prepared and changed from serum-free CM of this cell line. The heterologous protein was purified. Analysis by reduced SDS-PAGE revealed that purified sCD1 4_{(7-10) A}Moved to a position with an apparent molecular weight of 55,000 (data not shown) . Amino acids 7-10 have been replaced with alanine residues by N-terminal sequencing. And confirmed.                               Example 3 mAb 3C10 does not recognize purified sCD14 _{(7-10) A}   Using BIAcore real-time analysis again, mAb 3C10 was purified. CD14_{(7-10) A}It was determined whether it could be bound. FIG. 4 shows that sCD14_1-348Is Recognizes immobilized 3C10 and causes an increase of 1800 RU 2 minutes after washing (T = 30 RU of sensorgram before HCl injection at 0 and sCD14 at T = 0_1-348Injection (Compare it before) and confirmed previous observations (Juan, T.S.-C. et al., J. Biol. .Chem. 270, 1382-1387 (1995)). However, purified sCD14_{(7-10) A}Is 3C10 Can not be recognized and can be observed when irrelevant proteins such as bovine serum albumin are injected. Only a small RU change similar to the RU change (The RU of the sensorgram after the second wash at T = 750, T = 0 SCD14_1-348Amino acids 7-10 are mAb 3C 10 required for binding.                               Example 4 sCD14 _{(7-10) A} shows reduced ability to mediate cellular responses to LPS   To evaluate the results of mutating 7-10 residues of sCD14, two previous (Hailman, E. et al., J. Exp. Med. 179, 269-277 (1994). ); Juan, T.S.-C. Et al., J. Biol. Chem. 270, 1382-1387 (1995); Frey, E.A. et al., J. Exp.Med. 176, 1665-1671 (1992)), sCD14_{(7-10) A}Biological activity was measured. Most First, sCD14 enables U373 cells to respond to LPS_{(7-10) A}Noh The force was tested. Low concentrations of sCD14 at 5 ng / mL in the presence of LPS_1-348 Addition enabled strong IL-6 production (FIG. 5A). In contrast, sCD14_{(7 -10) A} Showed a greatly reduced ability to respond and sCD14_1-348Response similar to Required about 10-fold protein (FIG. 5A).   Also, sCD14_{(7-10) A}Is LPS-induced adhesion of PMN to fibrinogen It was tested whether it was possible. FIG. 5B shows that sCD14_1-348100ng / mL enabled strong adhesion response of PMN to smooth LPS and rLBP Indicates that However, 10,000 ng / mL of sCD14_{(7-10) A}Was added Even very few responses were seen. These findings suggest that amino acids Confirm that regions 7-10 are required for sCD14 biological activity.                               Example 5 sCD14 _{(7-10) A} has the ability to activate the transcription factor NF-κB in the presence of LPS. Show decline   LPS and sCD14-mediated activation of cells requires activation of transcription factors such as NF-κB. (Sen, R. and Baltimore, D. Cell 47, 921-928 ( 1986); Lee, J.D., et al., J. Exp. Med. 175, 1697-1705 (1992); Baqasra, D. et al., Pr. oc.Natl.Acad.Sci.U.S.A. 89, 6285-6289 (1992)). sCD14_{(7-10) A}Mutation Wild-type or modified to assess downstream signaling. Processed with variant sCD14 NF-κB activation in cultured U373 cells was tested. Absence of LPS or sCD14 In the presence of U373 cells, endogenous NF forms a complex with the labeled NF-κB probe. It has -κB (complex 1, FIG. 6, lane 1). LPS only or sCD14_{1-34 8} Stimulation with only a slight increase in NF-κB complex 1 and new NF-κB Slight induction of the complex was induced (complex 2, FIG. 6, lanes 2 and 3), but sC D14_1-348And the addition of LPS greatly induced both complexes of NF-κB ( FIG. 6, lanes 1 and 4 are compared). Both complexes 1 and 2 are NF-κB specific. Because a 100-fold excess of preincubated with U373 cell extract Unlabeled NF-κB oligonucleotide prevented the formation of both complexes. (Data not shown). sCD14_{(7-10) A}Stimulation of U373 cells with LPS Caused only 5% of NF-κB activation by quantification by gel scanning (FIG. 6, laser 6). By comparison, a mutant that does not bind to LPS (sCD14_Δ57-64) [Note: Δ57-64 means deletion of amino acids 57-64] Intensely, the NF-κB complex could not be activated even in the presence of LPS (FIG. 6). , Lane 8). These days SCD14_{(7-10) A}Defects are observed at the level of the transcription factor NF-κB. And NF-κB activation is an early event in signal transduction (Gril li, M. et al., Int. Rev. Cytol. 143, 1-62 (1993), these data indicate that sCD14_{(7- 10) A} Indicates that the signal cannot be transmitted.                               Example 6 sCD14 _{(7-10) A} forms a stable complex with LPS   sCD14_{(7-10) A}Decreased signal transduction is due to a defect in binding to LPS. It can be due to a fall. sCD14_{(7-10) A}Directly evaluates whether or not binds to LPS normally. To evaluate, sCD14_1-348Or sCD14_{(7-10) A}When^ThreeStability with H-LPS A native PAGE assay was used to detect the complex. It has already been reported [Ha ilman, E .; J. et al. Exp. Med. 179, 269-277 (1994) )]_1-348The formation of a stable complex between LPS and LPS 30 minutes after the addition (FIG. 7A), the formation of the complex occurs when rLBP is added. SCD4 required for_1-348Was decreased (lane 2 in FIG. 7B). And lane 2 of FIG. 7A). This is because rLBP is sCD14 of LPS. The above observations of facilitating transfer to [Hailman, E .; And above) You. Interestingly, sCD14_{(7-10) A}Also in the absence of rLBP^ThreeH-LPS and Can form a stable complex, and the formation of this complex is also promoted by rLBP. (Compare lane 5 in FIG. 7B with lane 5 in FIG. 7A). These de Data is sCD14_{(7-10) A}Between LBP and LPS in vitro And that it can be done independently of LBP. Yes, sCD14_{(7-10) A}Decrease in biological activity of Not suggest that.                               Example 7 Inhibition of LPS-induced cell response by high concentrations of sCD14   sCD14_{(7-10) A}To further confirm that can bind LPS, SCD14 inhibits LPS-mediated cell activation Was used. In the first assay, fibrils of PMN induced by LPS SCD14 inhibits adhesion to linogen_{1- 348} Or sCD14_{(7-10) A}(Figure 8A). In this experiment, constant Increased concentrations of LPS and rLBP (1 → 100 μg / ml) of sCD14_1-348 Or sCD14_{(7-10) A}Incubated with Both proteins are LP S could be neutralized and inhibit LPS-induced PMN adhesion.   As shown for recombinant sCD14 expressed in baculovirus [ Haziot, A .; J. et al. Immunol. 152, 5868-5876 (1 994)], sCD14 mediated LPS-mediated TNF-α production in whole blood assays._{(7 -10) A} Was also examined whether it could be inhibited. In whole blood assays, increasing amounts of sCD1 4_1-348Or sCD14_{(7-10) A}Addition inhibited the production of TNF-α (FIG. 8). B), the addition of bovine serum albumin did not inhibit the production of TNF-α, The above findings (Haziot, A. et al., Supra) are supported. These days SCD14_{(7-10) A}But LPS and sCD14_1-348To interact with Proven.                         Discussion of Examples 1 to 7   In the above example, the epitope that neutralizes mAb 3C10 was sCD14 In the 7-14 amino acid region. Substitution of alanine residues in this region As a result, the binding between 3C10 and sCD14 was prevented. These data are 3C Ten epitopes are located within the first 152 amino acids of sCD14 and residues 57-6 Of the present inventors that the epitope is different from the epitope of MEM-18 [Juan , T .; S. -C. J. et al. Biol. Chem. 270, 1382-1387 ( 1995); Juan, T .; S. -C. J. et al. Bjol. Chem. 270, 5219-5224 (1995)]. 3C10 epitope is CD14 To help understand how it contributes to the function of sCD14_{(7 -10) A} Was purified, demonstrating that this protein has a very low ability to activate cells. The inability of the protein to promote NF-κB activation indicates that sCD14_{(7 -10) A} Cannot support LPS-mediated signaling.   sCD14_{(7-10) A}Deficiency in the signaling of Probably not because it could not bind properly or could not interact with LBP . Gel movement (FIG. 7A) and sCD14 as examined in two cell-based assays (FIG. 8)._{(7-10) A} Normally binds to LPS and rLBP is sCD14 of LPS._{(7-10) A}Move to Promote movement (FIG. 7B). These data indicate that 3C10 has sCD14 and LPS Of the present inventors that they normally bind to the complex of (Juan, TS-C) . Et al., Supra). In these experiments, other reports [Wright t, S. D. Et al., Science 249: 1431-1433 (1990); Viriyakosol, S.M. And Kirkland, T .; N. , J. et al. Biol . Chem. 270, 361-368 (1995)]. -LPS and sCD14 but not binding of LBP complex to cell surface CD14_{(7-1 0) A} Direct binding was determined.   sCD14_{(7-10) A}Binds normally to LPS, so sCD14_{(7-10) A}The sig It is thought that the null transmission defect is clearly shown on the cell membrane. The present inventor [Frey, E. FIG. A. J. et al. Exp. Med. 176,1665-1671 (1992)] And other researchers [Pugin, J .; Proc. Natl. Acad. Sci . U. S. A. , 90, 2744-2748 (1993); Haziot, A .; J. et al. Immunol . 151, 1500-1507 (1993); Arditi, M .; Et al., Infe ct. Immun. 61, 3149-3156 (1993)], LPS and / or Or a transmembrane protein that interacts with CD14 and transmits a signal to the cytoplasm Assumed existence. Thus, the interaction of sCD14 with this transmembrane component requires residue 7 -10 are considered essential. Alternatively, sCD14_{(7-10) A}Of the cell Transport of LPS to the lipid bilayer may be defective. Recently, the inventor has suggested that sCD14 Described LPS as HDL particles [Wurfel, M .; M. J. et al. Exp. Med. 18 1: 1743-1754 (1995)] and phospholipid vesicles (Wurfel, M .; M. And S.I. D. W. , While preparing the manuscript) Thus, residues 7-10 are essential for transporting bound LPS to the plasma membrane it is conceivable that.                               Example 8 Gram-positive bacterial cell components compete with LPS for binding to sCD14   FIG. aureus (SACE) phenol extract That the positive molecule can bind to sCD14 and compete with LPS for the binding site Prove that. Other data (not shown) show that SACE CD14-dependently Indicates a strong stimulation. The binding site currently defined on CD14 is Responses initiated by Gram-negative bacteria as well as those initiated by Gram-positive bacteria Response can also be involved.Abbreviation description   The abbreviations used in the above section are BCIP: 5-bromo-4-chloro. -3-indolyl phosphate-toluidine salt; BPI: bactericidal / permeability enhancer Protein; CHO: Chinese hamster ovary; CD: Circular dichroism; CM: Nara Medium; HBSS: Hanks balanced salt solution; IL-6: interleukin-6; LALF: Limulus anti-LPS factor; LBP: LPS binding tan Protein; LPS: lipopolysaccharide; NBT: p-nitroblue tetrazolium chloride PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffered saline; PM N: polymorphonuclear leukocyte; r: recombinant; RU: response unit; sCD14: soluble CD 14; ELTSA: Enzyme-linked immunosorbent assay.   Having concluded the description of the present invention, those skilled in the art will recognize that the present invention as described herein Obviously, many modifications and variations are possible without departing from the spirit and scope of the art. There will be.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), OA (BF, BJ, CF, CG , CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, U G), AM, AT, AU, BB, BG, BR, BY, C A, CH, CN, CZ, DE, DK, EE, ES, FI , GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, MD, M G, MN, MW, MX, NO, NZ, PL, PT, RO , RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, UZ, VN (72) Inventors Jiyuan, Shiao Qi             United States, California 93021,             Moore Park, Broadview Dry             B 11373 (72) Inventor Reikenstein, Henry S.             U.S.A., CALIFORNIA 73004,             Benjura, Reusan Street             9586 (72) Inventor Wright, Samiuel Day             United States, New York 10538,             Larchmont, Briar Close 2

Claims (1)

[Claims] 1. One of the amino acids at positions 1 to 6 of the sequence of FIG. 1 (where X ₁ -X ₄ are each independently selected from amino acids having Gly and C ₁ -C ₆ aliphatic side chains) Or a polypeptide comprising an amino acid sequence ending with one of the amino acids 152-348, or a physiologically acceptable salt thereof. 2. The polypeptide of claim 1, wherein the amino acid sequence comprises amino acids 1-152 of the sequence of FIG. 1. 3. The polypeptide of claim 1, wherein the amino acid sequence comprises amino acids 1 to 348 of the sequence of FIG. 1. 4. The polypeptide according to claim 1, wherein X ₁ -X ₄ are each independently selected from Gly, Ala, Val, Leu, Ile and Pro. 5. The polypeptide of claim 1, wherein X ₁ -X ₄ are each Ala. 6. The polypeptide of claim 1, wherein X ₁ -X ₄ are each amino acids in the L-configuration. 7. Wherein said sequence, of the sequence of amino acids 1-152 or and comprises 1 to 348 X ₁ -X ₄ is L-Ala Figures 1 polypeptide of claim 1. 8. A polypeptide comprising an amino acid sequence beginning with the amino acid at position 15 in the sequence of FIG. 1 and ending with one of the amino acids at positions 152 to 348, or a physiologically acceptable salt thereof. 9. The polypeptide of claim 9, wherein the amino acid sequence comprises amino acids 15-152 of the sequence of FIG. 1. 10. 9. The polypeptide of claim 8, wherein said amino acid sequence comprises amino acids 15 to 348 of the sequence of FIG. 11. A polynucleotide encoding the polypeptide according to claim 8. 12. The polynucleotide according to claim 11, which is DNA. 13. 9. A method of treating a patient in need of treatment for an inflammatory condition, comprising administering to the patient an effective amount of the polypeptide of claim 1 or claim 8. 14． The method according to claim 13, wherein the effective amount is 0.1 mg / kg to 100 mg / kg. 15. A pharmaceutical composition comprising a mixture of the peptide according to claim 1 or 8 and a pharmaceutically acceptable carrier thereof.