EP0793677A1 - Polypeptides cd14 anti-inflammatoires - Google Patents

Polypeptides cd14 anti-inflammatoires

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Publication number
EP0793677A1
EP0793677A1 EP95944577A EP95944577A EP0793677A1 EP 0793677 A1 EP0793677 A1 EP 0793677A1 EP 95944577 A EP95944577 A EP 95944577A EP 95944577 A EP95944577 A EP 95944577A EP 0793677 A1 EP0793677 A1 EP 0793677A1
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EP
European Patent Office
Prior art keywords
leu
ala
pro
val
ser
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95944577A
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German (de)
English (en)
Inventor
Shao Chieh Juan
Henri S. Lichenstein
Samuel D. Wright
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Rockefeller University
Amgen Inc
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Rockefeller University
Amgen Inc
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Priority claimed from US08/366,953 external-priority patent/US5766593A/en
Application filed by Rockefeller University, Amgen Inc filed Critical Rockefeller University
Publication of EP0793677A1 publication Critical patent/EP0793677A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field of polypeptides that have anti-inflammatory properties.
  • Amino acids 7 to 10 of CD14 have been found to contain an important domain enabling inflammatory responses in cells, including IL-6 production.
  • the polypeptides of this invention were based on replacing amino acids 7 - 10 in the soluble form of CD14 with different amino acids.
  • Other polypeptides of this invention are missing the first 14 amino acids of soluble CD14.
  • the polypeptides of the invention may be used to treat inflammatory conditions, such as sepsis.
  • endotoxins that are typically present on the outer membrane of all gram-negative bacteria are among the most studied and best understood sepsis-inducing substances. While the precise chemical structures of LPS molecules obtained from different bacteria may vary in a species-specific fashion, a region called the lipid A region is common to all LPS molecules. E. Rietschel et al., in Handbook of Endotoxins. 1: 187-214, eds. R. Proctor and E. Rietschel, Elsevier, Amsterdam (1984) . This lipid A region is responsible for many, if not all, of the LPS-dependent pathophysiologic changes that characterize sepsis.
  • LPS is believed to be a primary cause of death in humans afflicted with gram-negative sepsis, van
  • Sepsis is also caused by gram-positive bacteria. Bone, R.C. Arch. Intern. Med. f 154: 26-34 (1994) .
  • the activation of host cells can originate from gram-positive cell walls or purified cell components such as peptidoglycan and lipoteichoic acid. Such substances induce a similar pattern of inflammatory responses to those induced by LPS. Chin and Kostura, 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) .
  • LPS and gram-positive cell wall substances cause polymorphonuclear leukocytes, endothelial cells, and cells of the monocyte/macrophage lineage to rapidly produce and release a variety of cell products, including cytokines, which are capable of initiating, modulating or mediating humoral and cellular immune responses and processes.
  • cytokine alpha-cachectin or tumor necrosis factor (TNF- ⁇ )
  • TNF- ⁇ tumor necrosis factor
  • Intravenous injection of LPS into experimental animals and man produces a rapid, transient release of TNF- ⁇ .
  • Pretreatment of animals with anti-TNF- ⁇ antibodies can modulate septic shock. Beutler et al., Science, 229: 869, (1985); Mathison et al., J. Clin. Invest. 81; 1925 (1988).
  • CD14 is a 55-kD glycoprotein expressed strongly on the surface of monocytes and macrophages, and weakly on the surface of granulocytes, such as neutrophils.
  • CD14 is linked by a cleavable glycosyl phosphatidyl inositol tail [A. Haziot et al., J. Immunol.
  • CD14 mediates responses by binding to LPS.
  • LBP acts in a catalytic fashion, with one molecule of LBP transferring hundreds of LPS molecules to hundreds of CD14 molecules.
  • Other experiments have shown that cell activation can also be induced by interaction of CD14 with components of gram-positive bacteria such as B. subtilis, S. aureus, and 5. mitus (Pugin et al., Immunity 1: 509-516 (1994) .
  • CD14 is a receptor which recognizes a wide variety of bacterial structures. Interaction of CD14 with these structures initiates host inflammatory responses.
  • CD14 has also been shown to exist as a soluble protein found in normal sera or urine of nephrotic patients. Recent evidence has shown that sCD14 enables LPS-dependent responses in cells which lack membrane CD14, i.e., endothelial cells and epithelial cells. In these cells types, sCD14 in conjunction with LPS promotes inflammatory cytokine release and upregulation of adhesion molecules.
  • sCD14 like CD14 neutralizing monoclonal antibodies, could be useful in preventing LPS interactions in mCD14.
  • the utility of sCD14 to treat LPS-mediated inflammatory disorders is limited by its other property of eliciting inflammatory cytokines in endothelial cells.
  • a sCD14 molecule which retained its ability to bind LPS, yet did not activate endothelial cells should have superior properties in treating inflammation.
  • Monoclonal antibodies may be a useful tool to help identify domains in sCD14 required for cell activation.
  • mAbs MEM-18 and 3C10 recognize a sCD14 mutant truncated at amino acid 152, indicating that epitopes for these two mAbs are within the first 152 amino acids (Juan, T. S. -C, et al. J. Biol. Chem. 270, 1382-1387 (1995)).
  • mAb 3C10 defines another functional domain of CD14. This mAb appears to recognize a different region from that of MEM-18 (Juan, T. S. -C et al. J. Biol. Chem. 270, 1382-1387 (1995)). Binding of monoclonal antibody 3C10 to sCD14 does not affect LPS binding to sCD14 (Juan, T. S. -C, et al. _____ Biol. Chem. 270, 5219-5224 (1995)), suggesting that this epitope may be involved in a cellular function other than LPS binding.
  • diseases include ARDS, septic shock, acute pancreatitis, acute and chronic liver failure, intestinal or liver transplantation, inflammatory bowel disease, graft vs. host disease in bone marrow transplantation and tuberculosis.
  • the present inventors have discovered a group of polypeptides that are capable of binding to lipopolysaccharide (LPS) , resulting in inhibition of the binding of LPS or gram-positive cell components to membrane CD14, thus reducing or eliminating
  • LPS lipopolysaccharide
  • CD14-mediated inflammatory responses As used herein, inhibition of binding of LPS also means inhibition of binding to gram-positive cell components. This group of polypeptides was designed by the inventors based on their important discovery of an LPS-binding domain in CD14.
  • polypeptides of this invention are based on substituting the naturally-occurring amino acids at positions 7-10 of CD14, preferably in a soluble form, with neutral amino acids, preferably those having either a hydrogen or C1-C6 alkyl side chain.
  • the polypeptides of this invention also include those that begin at amino acid 15 of FIG. 1.
  • "soluble" CD14 (SCD14) is a molecule selected from sequences starting at an amino acid of from 1 through 6 and ending at an amino acid of from 152 through 348.
  • Native human CD14 has as X 1 -X4. Glu-Leu-Asp-Asp in FIG.l.
  • the polypeptide is substituted with alanine at each of positions 7 through 10.
  • polypeptides of this invention are capable of binding to LPS, thereby preventing further binding of microbial cell components to membrane CD14. If microbial cell interaction with membrane CD14 is prevented, the cascade of events leading to inflammation, and especially sepsis, are reduced or prevented. Therefore, the polypeptides of this invention have anti-inflammatory properties.
  • the evidence provided herein indicates, inter alia, that a polypeptide having the region from amino acids 7 to 10 in CD14 replaced with alanine residues or having amino acids 1 to 14 deleted from soluble CD14, binds LPS but mediates a substantially reduced cellular inflammatory response, such as production of the cytokine IL-6 in response to LPS, as compared to native CD14.
  • a polypeptide having the region from amino acids 7 to 10 in CD14 replaced with alanine residues or having amino acids 1 to 14 deleted from soluble CD14 binds LPS but mediates a substantially reduced cellular inflammatory response, such as production of the cytokine IL-6 in response to LPS, as compared to native CD14.
  • we identify the epitope of 3C10 by making a series of site-directed alanine substitution mutants in sCD14. We show that the region between amino acids 7 and 14 are required for 3C10 binding.
  • the peptides and polypeptides of this invention may be prepared by (a) standard synthetic methods, (b) derivation from CD14, (c) recombinant methods, (d) a combination of one or more of (a) - (c) , or other methods of preparing polypeptides.
  • polypeptides of this invention may be used for therapeutic or prophylactic purposes by incorporating them into appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other mammal) in need thereof.
  • FIG. 1 shows a schematic map of soluble human CD14 having 348 amino acids; amino acids 7 - 10 (shown as X1-X4) are Glu-Leu-Asp-Asp in native mature human CD14.
  • amino acids 7 - 10 may be any of the amino acids described herein.
  • FIG.2 shows the sequence and expression of sCD14 alanine-substitution mutants.
  • FIG. 3 shows the results of a BIAcore analysis of the monoclonal antibody 3C10 binding to alanine substitution mutants of sCD14.
  • Conditioned media CM
  • MOCK no DNA
  • sCD1 ⁇ _3 4 8 sCD14 mutants four days after electroporation. All CM were analyzed for their ability to bind the antibody 3C10 as described in the Examples section herein.
  • Relative response units RRU
  • FIG. 4 shows that mAb 3C10 does not recognize purified SCD1 ( 7 _IO) A .
  • Immobilization of mAb 3C10 to a sensor chip has been described (Juan, T. S. -C, et al. J. Biol . Chem. 270 r 1382-1387 (1995)).
  • lO ⁇ g/ml sCD1 ⁇ _348 or SCD1 ( _ I O) A was used for injection.
  • Injection of solutions at various "Steps" are marked on the sensorgram.
  • “Wash” indicates a washing step using HBS buffer as described in the Example Section. The experiments were performed three times and the results of one experiment are shown.
  • FIG. 5 shows that SCD14 7 _ 10 ) A is defective in enabling cellular responses to LPS.
  • SCD14 (7 _ I O) A has reduced ability to stimulate IL-6 production by U373 cells.
  • U373 cells were treated with various concentrations of sCD14 1 _ 348 or SCD14 (7 _ IO ) A in the presence or absence of LPS (20 ng/ml) for 24 h.
  • IL-6 levels were determined as described (Juan, T. S. -C, et al. J. Biol. Chem. 270, 1382-1387 (1995)). Data presented are means ⁇ standard deviations from four readings in an experiment repeated 3 times.
  • sCD14 ⁇ _ 348 but not SCD14( 7 _ IO ) A mediates responses of PMN to LPS and LBP. Freshly isolated PMN were incubated with "smooth" LPS (E. coli 0111:B4 30 ng/ml), rLBP (1 ⁇ g/ml) , and the indicated concentrations of sCD14 ⁇ _34 ⁇ or SCD14( 7 _IO)A f r 10 min at 37°C Cells were washed and adhesion to fibrinogen-coated wells was measured (Hailman, E., et al. J. Exp. Med. 179, 269-277 (1994),25). Error bars indicate standard deviations of triplicate determinations. FIG.
  • SCD14 (7 _ 10 )A does not activate NF-KB.
  • Whole cell extracts of U373 cells with various treatments (lane 1, control; lane 2, LPS; lane 3, sCD14 1 _ 3 4 8 ; lane 4, SCD14X- 34 8 and LPS; lane 5, SCD14 (7 _ 10 )A; lane 6, SCD14( _IO)A and LPS; lane 7, s D14 ⁇ 51 - 6 ) ; and lane 8, SCD14 ⁇ (5 7 _ 6 ) and LPS) were obtained and binding of proteins to the labeled NF-KB oligonucleotide was performed as described in the Example Section.
  • Complexes of NF-KB were resolved on a native-4.5% polyacrylamide gel. After electrophoresis, the gel was dried and exposed to X-ray film for 16 h. Complexes of labeled probe and NF-KB are indicated.
  • FIG. 7 shows that sCD14 (7 _ 10)A forms stable complexes with 3 H-LPS.
  • Various concentrations of SCD14 X - 348 (lanes 2-4) or SCD14 (7 _ I0 ) A (lanes 5-7) were incubated with 3 ⁇ g/ml 3H-LPS in the absence (A) or presence of 16.7 nM rLBP (B) as described in the Example Section.
  • Lane 1 contains LPS in the absence of additional protein. Mixtures were run on 4-20% native polyacrylamide gels and processed for fluorography.
  • FIG. 8 shows inhibition of LPS-induced cellular responses by sCD14( 7 -I O )A> A - Inhibition of LPS-induced PMN adhesion by SCD14 (7 _ 10 )A> Rough LPS
  • Fraction of TNF- ⁇ production refers to the ratio of TNF- ⁇ produced in the presence of exogenous protein divided by TNF- ⁇ produced in the absence of added protein. Error bars are standard deviations from six readings.
  • FIG. 9 shows that gram positive cell components compete with LPS for binding to sCD14.
  • 3 H-LPS (1 ⁇ g/ml) and sCD14 (50 ⁇ g/ml) were incubated alone (lane 1) or with LPS (lane 2) or with Staphylococcus aureus crude extract (SACE) (lanes 3-6) at 37° C for 17 hours in PBS with 1 mM EDTA.
  • SACE Staphylococcus aureus crude extract
  • FIG. 1 shows a map of human CD14, including the LPS binding and IL-6 inducing region, corresponding to amino acids 7 to 10.
  • polypeptides of this invention are based on substituting amino acids at positions 7 - 10, inclusive, in soluble CD14, with amino acids that are different from those in the native molecule or deleting the first 14 amino acids in soluble CD14.
  • amino acids 7 - 10 in the polypeptides that contain substituted amino acids are different from Glu- Leu-Asp-Asp.
  • the substituted amino acids are neutral amino acids.
  • halogen e.g., Cl, Br, I
  • R alkyl having 1 to 6 carbon atoms
  • the specific amino acids substituted at amino acids 7 - 10 are relatively unimportant as long as the resulting polypeptide is capable of binding LPS and/or inhibiting release of inflammatory mediators in monocytes and PMNs while at the same time possessing a diminished ability (compared to wild type sCD14) to induce an inflammatory response in endothelial cells and epithelial cells.
  • polypeptides of this invention that have the initial 14 amino acids deleted from soluble CD14 end at an amino acid in sCD14 of from 152 to 348, inclusive.
  • Preferred deletion-type peptides are amino acids 15-152 and 15-348 of FIG. 1.
  • Soluble CD14 means polypeptides that correspond to amino acids of from positions 1 through 6 to positions 152 through 348, of FIG. 1. Note that in all cases herein the numbering of amino acids used herein corresponds to the amino acids sequence of FIG. 1. For example, even if the particular soluble CD14 starts with the second through sixth amino acid as set forth in FIG. 1, the amino acids to be substituted are those corresponding to amino acids 7 - 10 of the full sequence of FIG. 1.
  • sCD14 or sCD14 ⁇ - 348 the molecule depicted in FIG. 1 will be referred to as sCD14 or sCD14 ⁇ - 348 .
  • Other examples of soluble CD14 will be named by providing the beginning and ending amino acids based on the numbering scheme of FIG. 1; e.g., sCD14 ⁇ _i52,* sCD14 2 -i 52 ; sCD14i5_ ⁇ 52 ; sCD14 15 _3 48 , etc.
  • Polypeptides having amino acids substituted for those set forth in FIG.l will be named as follows: sCD14 ⁇ - 34 8 having alanine in place of amino acids 7 - 10 in FIG. 1 is named sCD14 ⁇ -34 ⁇ (7-i0)A.
  • sCD14 ⁇ _ 34 ⁇ having an alanine at positions 7 and 9-10, and a glycine at position 8 is named sCD14 ⁇ _34 ⁇ (7, 9, 10)A(8)G.
  • polypeptides of this invention are set forth below:
  • the present invention also encompasses physiologically acceptable salts of the polypeptides disclosed herein.
  • one or more D or L amino acids may be included; however, it is preferred that all of the amino acids are of the L stereochemistry.
  • the polypeptides of this invention are expected to have the ability to reduce inflammatory responses in cells as compared to native sCD14. Reduction in inflammation may conveniently be measured by examining IL-6 production by such cells using, e.g., the method described in Example 4 below.
  • the amount of IL-6 reduction, as compared to native sCD14 will be at least 5-fold, particularly preferably, at least 10-fold.
  • amino acids may be chemically derivatized as long as LPS binding coupled with reduced ability to induce IL-6 (or related cytokines) is retained.
  • chemical derivatives of the present polypeptides are included within the scope of the term “polypeptide” as used herein. These chemical derivatives contain additional chemical moieties not part of the Xi - X 4 amino acid substituted polypeptides.
  • Covalent modifications of the polypeptides are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides-. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • Carboxyl side groups are selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as l-cyclohexyl-3-(2-morpholinyl-(4- ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide.
  • carbodiimides R'-N-C-N-R'
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues.
  • these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular carriers.
  • cross-linking agents include, e.g., 1, 1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysucc broadlymide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3' - dithiobis (succinimidylpropionate) , and bifunctional maleimides such as bis-N-maleimido-1,8-octane.
  • Derivatizing agents such as methyl-3-[ (p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • the activity of the polypeptide variant can be screened in a suitable screening assay for the desired characteristic.
  • Biological activity is screened in an appropriate bioassay, as described herein.
  • binding of LPS to CD14 (or a polypeptide of this invention) may be measured in a standard competitive binding assay.
  • Activity to reduce cellular inflammatory responses may be measured in terms of reduction of IL-6 production by cells (e.g., U373 cells) as described herein.
  • Such derivatized moieties may improve the solubility, absorption, biological half life, and the like.
  • the moieties may alternatively eliminate or attenuate any undesirable side effect of the protein and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed.. Mack Publishing Co., Easton, Pa. (1980) .
  • the polypeptides of the invention may also be covalently or noncovalently associated with a carrier molecule, such as a polypeptide or non-CD14 protein, a linear polymer (such as polyethylene glycol, polylysine, etc), a branched-chain polymer (see, for example, U.S.
  • polypeptides of this invention are expected to have the ability to bind to LPS. This binding renders LPS unable to bind to membrane CD14 (mCD14) on macrophages and therefore produces an anti- inflammatory response in a mammal. Additionally, the polypeptides of this invention have reduced ability to trigger an inflammatory response in cells lacking mCD14 such as endothelial and epithelial cells. They are also expected to bind to cellular components of gram positive cells that cause inflammation (analogous to LPS; however, the structure(s) in gram positive bacteria that cause inflammatory responses to cells is (are) not yet known) .
  • Binding to LPS means that in a standard competition assay, the polypeptide is capable of inhibiting 50% binding of CD14 to LPS between 1 mM and 1 nM, preferably 100 ⁇ m to 10 nM (IC50 values) .
  • a standard binding assay may be carried out as is well known in the art.
  • polypeptides of this invention may be made in a variety of ways. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield, in Chem. Polypeptides. pp. 335-61 (Katsoyannis and Panayotis eds. 1973); Merrifield, ____ Am. Chem. Soc. 85, 2149 (1963); Davis et al., BiPChem. Int'l. 10, 394-414 (1985); Stewart and Young, £aJi--i
  • the polypeptides are made in transformed host cells using recombinant DNA techniques.
  • a recombinant DNA molecule coding for the polypeptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the polypeptides could be excised from DNA using suitable restriction enzymes.
  • the invention also includes a vector capable of expressing the peptides in an appropriate host.
  • the vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known.
  • Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.
  • the resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art.
  • Any of a large number of available and well- known host cells may be used in the practice of this invention.
  • the selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity to it of the peptides encoded for by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence.
  • useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.
  • bacteria such as E. coli sp.
  • yeast such as Saccharomyces sp.
  • other fungi insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.
  • the transformed host is cultured under conventional fermentation conditions so that the desired peptides are expressed.
  • Such fermentation conditions are well known in the art.
  • polypeptides are purified from the culture. These purification methods are also well known in the art.
  • polypeptides of this invention may be used in any of a number of situations where LPS/gram positive cell component binding is required.
  • the polypeptides may be used for inflammatory bowel disease, acute and chronic liver failure, graft vs. host disease (bone marrow transplant) , intestinal or liver transplant,
  • Septic shock is a particularly preferred target condition.
  • novel polypeptides are useful for the prophylaxis or treatment of septic shock in mammals, including humans, at doses of about 0.1 to 100 mg/kg of body weight, preferably at a level of about 1 to 50 mg/kg of body weight, and the amount may be administered, e.g., in divided doses on daily basis.
  • the polypeptides may be administered prophylactically to patients who may be exposed to or have been exposed to organisms which may cause septic shock or to detoxify LPS (bacterial endotoxins) by the use of the same dose set forth above in vivo; in vitro detoxification or prevention of endotoxin contamination may be carried out at a level which is effective to achieve the desired result.
  • LPS bacterial endotoxins
  • the amount may be based on routine experimentation based on the premise that about 1 mole of endotoxin is bound by 1 mole of polypeptide.
  • the particular dose of a particular polypeptide may be varied within or without the range that is specified herein depending on the particular application or severity of a disease and the condition of the host. Those who are skilled in the art may ascertain the proper dose using standard procedures.
  • compositions of the present invention may be administered by any means that achieve their intended purpose.
  • administration may be by parenteral routes, including subcutaneous, intravenous ⁇ intradermal, intramuscular, intraperitoneal, intrathecal, transdermal, or buccal routes.
  • parenteral routes including subcutaneous, intravenous ⁇ intradermal, intramuscular, intraperitoneal, intrathecal, transdermal, or buccal routes.
  • administration may be by the oral or rectal route.
  • the pharmaceutical compositions can be administered parenterally by bolus injection or by gradual perfusion over time.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations particularly those which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.1 to about 99 percent, preferably from about 25-85 percent, of active compound(s), together with the excipient.
  • Suitable excipients are, in particular, fillers such as sugars, such as lactose, sucrose, mannitol, or sorbitol; cellulose preparations and/or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; as well as binders such as starch paste made using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethyl cellulose, and/or polyvinylpyrrolidone.
  • fillers such as sugars, such as lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations and/or calcium phosphates such as tricalcium phosphate or calcium hydrogen phosphate
  • binders such as starch paste made using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
  • disintegrating agents may also be added, such as the above- mentioned starches as well as carboxymethyl starch, cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries which can be used in the compositions according to the present invention include flow-regulating agents and lubricants such as silica, talc, stearic acid or salts thereof, a detergent such as Triton, and/or polyethylene glycol.
  • Reagents Recombinant soluble CD14 (rsCD14) and recombinant LBP (rLBP) were constructed and purified as described (Hailman, E., et al. J. Exp. Med. 179, 269-277 (1994)). Concentrations of all purified proteins were determined with a Micro BCA protein kit (Pierce, Rockford, IL) according to manufacturer's specification. Since full-length rsCD14 terminates at position 348 of the mature protein (Hailman, E., et al. J. Exp. Med. 179, 269-277 (1994)), we herein refer it as sCD14 1 - 348 .
  • the anti-CD14 mAb 3C10 was purified by chromatography on Protein G from the conditioned medium (CM) of a cell line from American Type Culture Collection (ATCC TIB 228) .
  • CM conditioned medium
  • ATCC TIB 2208 Rabbit polyclonal anti-human CD14 antiserum was prepared as described (Juan, T. S. -C, et al. J. Biol. Chem. 270, 1382-1387 (1995)).
  • Rough LPS Salmonella minnesota R60 or Re595
  • smooth LPS E. coli 0111:B4 or Salmonella minnesota wild-type
  • Enzymes for DNA manipulation were purchased from Boehringer Mannheim (Indianapolis, IN) .
  • FIG. 2 summarizes the names and the amino acid residues substituted in each mutant.
  • the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA) was used as previously described (Juan, T. S. -C, et al. J. Biol. Chem. 270. 5219-5224 (1995)) to generate cDNAs encoding alanine-substitution mutants of sCD14 cloned in a mammalian expression vector.
  • the primers used for each mutant are as follows:
  • mutant sCD14 proteins To express mutant sCD14 proteins, mammalian expression vectors containing mutant sCD14 cDNAs were introduced into COS-7 (ATCC CRL 1651) cells by electroporation. Conditions for electroporation and generation of serum-free CM from transfected COS-7 cells were as described (Juan, T. S. -C, et al. J. Biol. Chem. 270, 1382-1387 (1995)). Expression of mutant sCD14 was analyzed by Western blot using anti-CD14 polyclonal antibody.
  • CM5 sensor chips CM5 sensor chips
  • amine coupling kit purchased from Pharmacia Biosensor (Piscataway, NJ) . Briefly, mAb 3C10 (200 ⁇ g/ml in 20 mM sodium acetate, pH 3.4) was immobilized to a CM5 sensor chip by amine coupling according to manufacturer's specifications.
  • Step 1 COS-7 CM for 2 min and Step 2, HBS buffer [10 mM N-2-hydroxyethylpiperazine-2V I -2-ethanesulfonic acid, pH 7.5, 0.15 M NaCl, 3.4 mM EDTA, 0.005% (V/V) surfactant P20 (Pharmacia Biosensor) ] for 2 min.
  • HBS buffer 10 mM N-2-hydroxyethylpiperazine-2V I -2-ethanesulfonic acid, pH 7.5, 0.15 M NaCl, 3.4 mM EDTA, 0.005% (V/V) surfactant P20 (Pharmacia Biosensor)
  • 10 mM HC1 solution was injected for 2 min. Injection was performed at a rate of 5 ⁇ l/min.
  • RRU response unit
  • SCD14 (7 _ 10jA Purification of SCD14 (7 _ 10jA .
  • the expression vector containing the cDNA encoding sCD14 (7 _ 10)A was stably transfected into Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase as described (Hailman, E., et al. J. Exp. Med. 179, 269-277 (1994)). A single clone was grown without serum to generate CM containing sCD14( 7 - ⁇ o) A - Mutant protein was purified exactly as described (Juan, T. S. -C, et al. J. Biol. Chem.
  • U373 cells ATCC HTB17, Rockville, MD
  • activation by purified sCD14 preparations and quantitation of IL-6 were performed exactly as described (Juan, T. S. -C, et al. J. Biol. Chem. 270, 1382-1387 (1995)). Briefly, mixtures of sCD14 ⁇ - 3 4 8 or SCD14( 7 _I 0 )A and LPS were added to monolayers of U373 cells in serum—free medium and incubated for 24 h. IL-6 in the supernatant was then measured by ELISA.
  • PMN Polymorphonuclear Leukocytes
  • Extracts were quickly frozen in liquid nitrogen and stored at -80°C. Protein concentration of the whole cell extracts were determined by micro BCA assay and ranged between 1.5-2 ⁇ g/ ⁇ l. For examining the NF-KB complexes, we performed electrophoretic mobility shift assays. Two oligonucleotides:
  • 5'-CATGGAGGGACTTTCCGCTGGGGACTTTCCAGC-3' and 5'-CATGGCTGGAAAGTCCCCAGCGGAAAGTCCCTC-3' were annealed to generate a double-stranded DNA containing the NF-KB binding site of human immunodeficient virus long terminal repeat promoter (Nabel, G. and Baltimore, D. Nature 326, 711-713
  • the reactions were incubated in a 30°C water bath for 30 min and complexes were resolved in a native 4.5% polyacrylamide gel using 0.5 X TBE (50 mM Tris-HCl, pH 8.0, 45 mM boric acid, and 5 mM EDTA) at 30 mA for 2 h.
  • the gel was then vacuum-dried at 80°C for 1 h and exposed to Kodak X-ray film for 20 h.
  • 100 X molar excess of unlabelled NF-KB probe was pre-incubated for 10 min before addition of radioactive probe.
  • sCD14 ⁇ _ 348 or sCD14 (7 _ 10)A were incubated at various concentrations (0, 101, 303, and 909 nM) with 3 ⁇ g/ml of 3 H-LPS prepared from E. coli K12 strain LCD25 (List Biological Laboratories) in the presence or absence of 16.7 nM rLBP.
  • the reaction was incubated at 37°C for 30 min and then electrophoresed on native 4-20% polyacrylamide gels. Gels were prepared for fluorography as previously described (Hailman, E., et al. J. Exp. Med. 179, 269-277 (1994)).
  • 3C10 is a mAb that recognizes the N-terminal 152 amino acid of CD14 (Juan, T. S. -C, et al. J. Biol. Chem. 270, 1382-1387 (1995)).
  • Previous experiments have shown that 3C10 neutralizes the activity of sCD14 ⁇ _ 34 8 (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.
  • SCD14 ( 7 - ⁇ o) A has Reduced Ability to Mediate Cellular Responses to LPS.
  • we used two previously described assays (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)) to measure sCD14( 7 _ ⁇ o)A bioactivity.
  • FIG. 5B shows that 100 ng/ml sCD14 ⁇ _ 348 enabled a strong adhesive response of PMN to smooth LPS and rLBP. However, very little response was seen even when 10,000 ng/ml SCD14( 7 _I O ) A was added.
  • sCD14 ( 7_IO ) A is Impaired in its Ability to Activate Transcription Factor NF-KB in the Presence of
  • FIG. 6, lane 1 Stimulation with LPS alone or sCD14 ⁇ _ 348 alone caused slight enhancement of NF-KB complex 1 and slight induction of a new NF-KB complex
  • U373 cells eliminated formation of both complexes (data not shown). Stimulation of U373 cells with sCD14( 7 _ 10 ) A and LPS caused only 5% of NF-kB activation as quantitated by gel scanning (FIG. 6, lane 6) . Comparatively, stimulation of U373 cells with a mutant which does not bind LPS (SCD14 ⁇ 57 - 64 ) [Note: ⁇ 57-64 means deletion of amino acids 57 - 64] failed to activate NF-KB complexes even in the presence of LPS (FIG. 6, lane 8) . These data indicate that a defect in SCD14 (7 _ !0)A is observed at the level at the transcription factor NF-KB.
  • SCD14( 7 _ IO > A could be due to a defect iii binding LPS.
  • SCD14 (7 _ IO)A binds LPS normally, we used a native PAGE assay to detect stable complexes between sCD14 1 _ 348 or sCD14( 7 _ ⁇ o) A and 3 H-LPS. As previously reported (Hailman, E., et al. J. EXP. Med. 179, 269-277 (1994)), formation of stable complexes between sCD14 1 _ 348 and LPS could be observed after 30 min of incubation (FIG.
  • LPS LPS
  • SCD14 X -348 or SCD14 (7 _I 0 ) A were tested for their ability to inhibit adhesion of PMN to fibrinogen induced by LPS (FIG. 8A) .
  • constant concentrations of LPS and rLBP were incubated with increasing amounts (from 1 to 100 ⁇ g/ml) of sCD14 ⁇ _ 3 8 or sCD14 (7 _ 10)A . Both proteins were capable of neutralizing LPS and inhibiting the adhesion of PMN induced by LPS.
  • SCD14( 7 _ I0 ) A binds LPS normally, as examined by gelshift (FIG. 7A) and two cell-based assays (FIG. 8) and rLBP facilitates transfer of LPS to sCD14( 7 _ 10 )A (FIG. 7B) .
  • FIG. 9 presents the evidence that a gram- positive molecule present in the phenol extract of S. aureus (SACE) can bind to sCD14 and compete with LPS for a binding site.
  • SACE phenol extract of S. aureus
  • Other data indicates that SACE strongly stimulates cells in a CD1 -dependent fashion.
  • the binding site(s) now defined on CD14 may be relevant not only to responses initiated by gram- negative but also by gram-positive bacteria.
  • BCIP 5-bromo-4-chloro-3-indoyl phosphate- toluidine salt
  • BPI bactericidal/permeability- increasing protein
  • CHO Chinese hamster ovary
  • CD circular dichroism
  • CM conditioned medium
  • HBSS Hank's balanced salt solution
  • IL-6 interleukin-6
  • LALF Limulus anti-LPS factor
  • LBP LPS-binding protein
  • LPS lipopolysaccharide
  • NBT p-nitro blue tetrazolium chloride
  • PAGE polyacrylamide gel electrophoresis
  • PBS phosphate-buffered saline
  • PMN polymorphonuclear leukocyte
  • r recombinant
  • RU response unit
  • sCD14 soluble CD14
  • ELISA enzyme linked immunosorbant assay.
  • Val Ser Ala Val Glu Val Glu lie His Ala Gly Gly Leu Asn Leu Glu 35 40 45
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:14:
  • MOLECULE TYPE cDNA

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Abstract

Cette invention concerne des polypeptides anti-inflammatoires comprenant des polypeptides solubles apparentés au CD14, lesquels possèdent en position 7 à 10 des acides aminés différents de la séquence d'origine, ou dans lesquels les acides aminés en position 1 à 14 ont été effacés.
EP95944577A 1994-12-30 1995-12-28 Polypeptides cd14 anti-inflammatoires Withdrawn EP0793677A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US08/366,953 US5766593A (en) 1994-12-30 1994-12-30 Anti-inflammatory CD14 peptides
US366953 1994-12-30
US484397 1995-06-07
US08/484,397 US5869055A (en) 1994-12-30 1995-06-07 Anti-inflammatory CD14 polypeptides
PCT/US1995/017095 WO1996020957A1 (fr) 1994-12-30 1995-12-28 Polypeptides cd14 anti-inflammatoires

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US6093693A (en) * 1996-11-18 2000-07-25 The Wellesley Hospital Foundation B cell activation
EP0911400A1 (fr) 1997-03-07 1999-04-28 Mochida Pharmaceutical Co., Ltd. Composes antisens diriges contre cd14
WO1999061468A2 (fr) * 1998-05-27 1999-12-02 Gemma Biotechnology Ltd. INDUCTION DE PROTEINES ET DE PEPTIDES ANTIBIOTIQUES AU MOYEN DE LA PROTEINE sCD14/LAIT (PROTEINE IMMUNOTROPHIQUE ASSOCIEE A LA LACTATION)
US7264967B2 (en) * 2000-11-22 2007-09-04 Mochida Pharmaceutical Co., Ltd. Anti-CD14 monoclonal antibody having effect of inhibiting CD14/TLR binding
JP4040666B2 (ja) * 2004-05-11 2008-01-30 持田製薬株式会社 新規可溶性ｃｄ１４抗原
CN101189334B (zh) 2005-06-03 2013-11-06 持田制药株式会社 抗cd14抗体融合蛋白质
US20100331239A1 (en) * 2008-01-29 2010-12-30 The Medical Research, Infrastructure And Health Services Cd14 and peptides thereof for protection of cells against cell death
CA2784919A1 (fr) 2010-01-26 2011-07-26 Banyan Biomarkers, Inc. Compositions et methodes liees a l'argininosuccinate synthetase
CN112745380B (zh) * 2021-01-22 2022-04-08 浙江辉肽生命健康科技有限公司 具有氨基酸结构raglqfpvgrvh的生物活性肽及其制备方法和应用

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CA2133758A1 (fr) * 1992-04-06 1993-10-14 Sanna M. Goyert Nouveau mode de traitement de la septicemie utilisant une forme soluble d'antigene myelomonocytaire cd14 recombinant

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AU4692396A (en) 1996-07-24

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