JPH09506258A - C5a receptor antagonist having substantially no agonist activity - Google Patents

Abstract

(57) SUMMARY Disclosed are polypeptide analogs of human C5a and their derivatives, and dimeric forms thereof, which are C5a receptor antagonists that exhibit substantially no anaphylatoxin or agonist activity. Also provided are DNA molecules encoding the peptides and methods for making the analogs. Pharmaceutical formulations containing C5a analogs are used therapeutically in the treatment of C5a-mediated diseases and inflammatory conditions, and to prevent or reduce inflammation caused by events that cause or exacerbate existing inflammatory conditions. Used prophylactically. Further disclosed are C5a analogs, derivatives thereof, and antibodies specific for their dimers that show substantially no cross-reactivity with human C5a. The antibodies are also used to detect or quantify circulating C5a analogs or derivatives thereof and to modify, eg neutralize, the in vivo activity of C5a receptor antagonists.

Description

Detailed Description of the Invention C5a receptor antagonist having substantially no agonist activityField of the invention   The present invention is directed to the field of immunology, and more particularly to complement-mediated diseases in mammals and It relates to the treatment of inflammatory conditions.Background of the Invention   Inflammation may destroy, dilute or enclose harmful substances and damaged tissue. Is a traumatically induced, localized protective event affecting the. It's complicated Sequence of events, such as dilation of arteries, capillaries and venules, and elevated blood vessels Includes permeability, accelerated blood flow, and exudation of fluids and plasma proteins. That After these steps, attachment of leukocytes to the vascular endothelium, followed by white blood into the tissue surrounding it. The inflow of spheres continues almost rapidly.   The complement system, the main immunological defense mechanism against foreign substances, is the inflammatory response. It has been shown to affect individual factors, including the answer. In general, the correction is , A set of proteins that act to eliminate microorganisms and antigens from tissues and blood Includes. This action expresses complement components only, or with antibodies, or complement receptors It is achieved by cooperating with cells that More specifically, the system is About 30 plasma proteins, their corresponding cellular receptors and some membrane regulatory factors Consisting of high quality. Kinoshita, Immunology Today, 12: 291-300 (1991). for example Activation of the complement system by, for example, the antigen-antibody complex or bacterial surface structures, Trigger a series of amplifications of proteolytic cleavage and protein assembly events in minutes Making This ultimately leads to the destruction and eventual elimination of foreign objects. Muller-Ebe rhard, Annu. Rev. Biochem. 57: 321-347 (1988).   Some biologically active peptides are produced by activation of the complement system. C5a, a glycoprotein containing 74 amino acids and having an Mr of about 11,000 The quality of C5 convertase is the N-terminal of C5, that is, the 5th component of complement. It is produced by protein degradation. Ntlsson et al., J. Immunol. 114 : 815-822 (1975). The biological properties of C5a are involved in both acute and chronic inflammatory processes Spread through a large number of cells and tissues. Hugli, CRC Crit. Rev. Immunol., 1: 321-366 (1981). Many of their properties are immunologically beneficial. C5a is , Have been found to mediate host defense mechanisms in response to various pathological conditions You. C5a is found in inflammatory responses such as smooth microcontraction, increased vascular permeability, and in human skin. When injected, wheals and redness develop, histamine release from mast cells, and Usually associated with triggering oxidative clustering and lysosomal enzyme release from polymorphonuclear leukocytes (PMNL) Are involved in a wide variety of specific biological functions. C5a is a female base, eosinophil Response from any circulating white blood cells, including monocytes and neutrophils Stimulates. Hugli, supra; Bautsch et al., Immunobiol. 185: 41-52 (1992). C 5a was further found to be a potential chemoattractant. Fernandez et al ., J. Immunol. 120: 109-115 (1978). This protein is found in PMNL It is a central stimulant for the attraction of vesicles to the site of inflammation.   Complement is directed against a suitable target, such as an invasive microorganism or tumor cell If beneficial, but improperly activated, it may have obvious pathogenic potential. Have. For example, anaphylatoxins, such as C5a, are inflammatory from several species. Diseases such as adult respiration Included as a causative or exacerbating factor in the etiology of difficult syndrome and rheumatoid arthritis Have been. Bautsch et al., Biochem. J. 288: 261-266 (1992); Haslett et  al., J. Immunol. 142: 3510-3517 (1989). In particular, the abnormalities of C5a in the organization Existence suffering from rheumatoid arthritis, osteoarthritis, psoriasis and non-cardiac pulmonary edema Have been detected by someone. Hammerschmidt, J. Amer. Med. Soc. 244: 199- (1980) . C5a has additional activities including recruitment and stimulation of inflammatory leukocytes and increased antibody production. Found to be the major inflammatory mediator produced by complementary activation by sex Mollison et al., Proc. Natl. Acad. Sci. Visit USA 86: 292-296 (1989) Teru.   The in vivo or pharmacological control of inflammation is postulated to depend on the regulation of chemotaxis. Three levels at which inhibition can occur are recognized. They are (1) for chemotactic stimulation Suppression of leukocyte response to; (2) prevention of chemotactic agent formation; and (3) inactivation of chemotactic agent It is sexualization. Furthermore, C5a is found in some human cells, such as neutrophils, eosinophils and By binding to a specific C5a receptor found on the membrane of monocyte-derived cells Since it exerts various functions, it inhibits C5a-mediated chemotaxis, and in particular C5a receptor The tagist is the attention of the person in charge.   U.S. Pat. No. 4,772,584 (Cleary et al.) Describes six C5a-terminal C-terminals. A group that inhibits C5a binding to PMNL by cleaving the mino acid peptide A shows a polypeptide isolated from streptococcus You. U.S. Pat.No. 4,692,511 (Hahn) shows a C5a blocking activity Essential core tetrapeptide Tyr-Asp-Gly-Ala (SEQ ID NO: 1) or Asp-Gly-Ala-Tyr ( SEQ ID NO: 2), or a polypeptide for C5a containing the core tripeptide Asp-Gly-Ala Teaching peptide receptor antagonists.   US Pat.No. 5,190,922, WO 90/09162 and 92/11858 (Abbo tt Laboritories) bind to the C5a receptor and, by definition, Disclosed are various oligopeptides that modify anaphylatoxin activity. But However, some of those molecules may retain significant agonitoto activity. It is shown. Mollison et al., “C5a Structural Reguiremants for Neufrop hil Receptor Intoraction, ”Progress in In flammation Research and Therap y, Brikhanser Verlag, Basel (1991), pp. 17-21; Kawai et al., J. Am. Med. C hem. 35: 220-223 (1992); Kawai et al., J. Med.Chem. 34: 2068-2071 (1991 ); And Or et al., J. Med. Chem. 35: 402-406 (1992). Therefore, Potential and therapeutically effective C5 receptor substantially lacking agonist activity There remains a strong need for body antagonists.Summary of the Invention   One aspect of the invention is a C5a receptor antagonist, and substantially A peptide analogue of human C5a that does not show nafilatoxin or agonist activity, And dimers of said analogues and derivatives. Polypeptide ( Ie, analogues and derivatives), DNA molecules, plasmids, vectors And a host cell transformed with the DNA molecule and a C5a antagonist. A method for making is also provided.   Pharmaceutical preparations containing C5a antagonists, their derivatives or dimers are C5a-mediated inflammatory conditions and diseases, and prevention to prevent such inflammation Conveniently used as a medicine.   Another aspect of the invention is that C5a which is substantially free of cross-reactivity with human C5a. Directed to antibodies specific for analogs and their derivatives. The antibody is the target ( Previously administered by the same) Detection or quantification of circulating C5a analogs and derivatives in It is also used to modify the activity of derivatives and derivatives in vivo, eg to neutralize.Brief description of the drawings   Figure 1 shows the coding for human C5a through oligonucleotide coupling. 2 is a flow chart showing the synthesis of the adult gene; and   FIG. 2 is a plasmid map of pB-6 / C5a.Description of preferred embodiments   The C5a polypeptide analogs of the invention are C5a polypeptides that have substantially no agonist activity. It is a receptor antagonist. The term "C5a receptor" refers to C5a, its degradation product C 5a-des Arg, and human antagonists, such as PM, to which the antagonist of interest binds It is understood in the art to refer to sites on the surface of NL and hemisphere cells. You. For example, US Pat. No. 5,177,190; and Oppermann et al., J. et al. Immuno l. 151 (7): 3785-3794 (1993). C5a is a carboxypeptidase Enzymatically converted to C5a-des Arg in human serum by a B-like enzyme, and human Is the main physiological end product in. Chenoweth et al., Mol. Immunol. 17 : 151-161 (1980).   The term "antagonist" means that the polypeptide disclosed in the present invention is an antagonist of C5a. It means that it is a hibitor. That is, they bind C5a to the C5a receptor Interfere with. I don't think it will be linked by any particular theory, Have found that C5a analogs compete with C5a for binding to the C5a receptor, resulting in Believe that it is a competitive inhibitor of.   The C5a analog antagonists of the present invention are detailed in Example 7. Seligmann et al., Agents and Actoons 21: 375-378 (198), described in detail. IC in the calcium elevation assay disclosed in 7)₅₀Can be quantified as This IC₅₀PMNL carrying C5a receptors following a challenge dose of 100 pM human C5a. As a concentration of C5a analogues that inhibits 50% of intracellular metabolism of calcium ions by Defined. The C5a receptor antagonists of the present invention were developed by Seligmann et al. Approximately 2.0 x 10 in the calcium elevation assay shown^-6IC not higher than M₅₀ Is shown.   The terms "substantially free of anaphylatoxin activity" or "substantially agonis "Has no activity," means that the C5a analog to the receptor (then the C5a receptor antago (Used interchangeably with nyst) results in an endogenous signal transduction event Or, ultimately, by the binding of C5a to its receptor. Phytotoxins Physiological responses normally associated with inflammation, such as phagocytic activation, normal Smooth muscle contraction, increased vascular permeability and inflammatory media such as histamine, prostag Landin, thromboxane, leukotriene, interleukin (IL) -I, IL -6 and means that it does not bring about the above events due to IL-8 overproduction . Hugli et al., CRC Crit. Rev. Immunol. 1: 321-326 (1981) and PCT WO 92/1 See 0205. A quantitative measurement of this property is also described in Example 7, Seligman. n et al., also obtained using the calcium elevation assay disclosed above. EC₅₀ Is a measure of agonist activity. For the purposes of this invention, EC₅₀Values are the same C5a analog Is the concentration of the C5a analog that produces 50% of the maximal response caused by. application Had at least about 8.0 x 10 in the same calcium elevation assay.^-7M and preferred At least about 3.0 x 10^-6Agonist activity of C5a analogs of the present invention up to concentrations of M Sex is not detected. The C5a analogs of the invention are derived from Seligmann's cal. Since no response can be detected in the sium elevation assay, at least about 8.0 x 10^{- 7} M, and preferably at least about 3.0 × 10^-6EC up to M5 C5a analogue concentration₅₀Before It is an analog that cannot be measured in the calcium elevation assay.   C5a is a 74-amino acid peptide whose sequence is described by Fernandez et al., J. Mol. Bio l. Chem. 253: 6955-6964 (1978). Deduced nucleotide Synthetic genes constructed on the basis of sequences are described in Mandecki et al., Proc. Natl. Acad . Sci. USA 82: 3543-3547 (1985) and US Pat. No. 4,937,189 (Davidow et al.). The amino acid sequence and the sequence of C5a disclosed by Fernandez And its corresponding synthetic nucleotide sequence disclosed by Davidow et al. 1 is shown.   Applicants have found that C- of human C5a (hereinafter used interchangeably with "C5a (1-74)") Mutagenesis of the terminal region, that is, the portion of the synthetic C5a gene encoding amino acids 64-74. A certain analog of human C5a, which was produced by eliciting It was surprisingly and surprisingly discovered that it has a property that has become That is, They exhibit outstanding antagonistic properties and show substantial agonist activity. Not shown. In particular, the C5a analogs of the invention include the C-terminal region of C5a (1-74): N'-Asn (64) -Ile (65) -Ser (66) -His (67) -Lys (68) -Asp (69) -Met (70) -Gln (71) -Le u (72) -Gly (73) -Arc (74) -C '(SEQ ID NO: 5) (amino acids 64-74 of C5a (1-74)) Is defined by two modifications or mutations of First, it is Gly (73) and Arg (74). By removing the residue, it is cleaved to at least Leu (72). Second, less Both systems have been replaced in that region, provided that the C of the polypeptide is The terminal amino acid (ie, C-terminal) is cysteine, and its C-terminal cis The thiol (SH) groups of thein are present in reduced form (ie have free thiol groups) , Or present in a form that can be converted spontaneously or easily converted into a free thiol group I do.   In a preferred embodiment, 2-6 of most C-terminal amino acids are C5a (1- 74) disconnected. Therefore, the 63 amino acid region at the N-terminus remains intact. If each of them is carried and only one cysteine is substituted, then the corresponding of each The embodiment can be designed as follows: C5a (1-74, Leu72Cys), C5a (1-71, Gln 71Cys), C5a (1-70, Met70Cys), C5a (1-69, Asp69Cys) and C5a (1-68, Lys6 8 Cys). In a more preferred embodiment, the C-terminal region is designed from the three former types. Cleaved to and including Met70, Gln71 or Leu72 corresponding to the embodiment. Sa Further preferred embodiments include C5a (1-71, Gln71Cys).   The C-terminal region is further truncated at Lys68 from the N-terminus, which is representative of C5 a (1-67, His67Cys), C5a (1-66, Ser66Cys), C5a (1-65, Ile65Cys) and C5a Corresponding to (1-64, Asn64Cys), except that the resulting C5a analog is Gonist property (about 2.0 × 10^-6IC not higher than M₅₀), And Anaphyra Toxin or agonist activity (at least 3.0 x 10^-6Up to C5a analogue concentration of M Non-measurable EC₅₀) Is not substantially indicated. Those skilled in the art will recognize that the "analog" of human C5a is C5 Understand that it does not include antibodies specific for sites on the a or C5a receptors. There will be.   Derivatives of human C5a analogs as described herein are included within the scope of the invention. Included. They are the N-terminal 63 amino acid region (amino acids of C5a (1-74) 1-63), point mutations, substitutions, additions and deletions (Carney et al., Protein Scien ce 2: 1391-1399 (1993)), and the C-terminal region thus mutated. Modifications such as additional amino acid substitutions in Type and degree of said modification Has substantially no agonist activity, as the resulting derivative is defined above. As long as it remains a non-C5a receptor agonist, it is generally unimportant. Tato For example, the Cys27 residue in the N-terminal region of C5a (1-74) adds complexity during regeneration. It may be changed, for example, to a serine residue to minimize it. Therefore, more preferable In an embodiment, the C5a analog derivative is designated C5a (1-17, Cys27Ser, Gly71Cys). Can be The N-terminus also encodes a C5a analog in various host cells. A methionine residue, either substituted or added, to allow expression of the gene. Can be changed to An example of additional modification of the C-terminal region is at position 67 in C5a (1-74). Substitution of a phenylalanine residue to replace conventional histidine. Therefore, the best In a better manner In addition, the C5a analog derivative is C5a (1-71, Cys27Ser, His67Phe, Gln71Cys) Is called.   C5a analogues of the invention (hereinafter referred to collectively as analogues and their derivatives) ) Can be prepared by many standard methods in the art. For example, they are It can be prepared by direct chemical synthesis. They are also available in suitable host cells , A DNA molecule that encodes a polypeptide, that is, prepared by expression of a synthetic gene. Can be Those DNA molecules that can be deduced from the amino acid sequences of C5a analogs are It can be prepared by the technique. Its DNA is from Narang, Tetrahedron 39: 3-22 (1983) and And E PA No. 146,785; Mondecki et al., Proc. Natl. Acad. Sci. USA 82: 3543 -3547 (1985), which discloses the chemical synthesis of the gene encoding C5a. Can be chemically synthesized as described above. Fragments of DNA molecules are chemically It can be prepared, which is then enzymatically linked together. Volumel, Chapter & of Ca rrent Protocols in Molecular Biology, Ausubel et al. (Eds.), Wiley, NY ( 1990).   The DNA encoding the C5a analog of the present invention also includes known synthetic or C5a-encoding DNA. Is disclosed in mutagenesis of natural genes, such as Fernandez, Mandecki and Davidson. Prepared by mutagenesis. Ausubel et al., Supra; Volume II, Chapter r 15 of Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Sp ring Harbor, NY (1989); and Mollison et al., Proc. Natl. Acad. Sci. USA  86: 292-296 (1989). In addition, DNA can be prepared by PCR techniques. You. PCR Protocols, Innis et al. (Eds.), Academic Press, San Diego, CA (19 See 90).   DNA molecules encoding the C5a analogs of the invention have known regulatory sequences, such as Motor, enhancer, 3'-untranslated sequence and 5 ' Operably linked to a translation sequence, such as a signal and leader sequence, and The gene is then expressed in a host cell capable of expressing the gene according to techniques known in the art. The quality is changed. The transformed host cell then encodes the antagonist It is cultured under suitable conditions for expression of the gene. A typical host cell is a prokaryote Objects such as E.I. Coli and Bacillus, for example B. Subtilis (B. subtilis); and eukaryotes, eg filamentous fungi, eg Aspergillus niger (Aspergillus niger; yeast, such as Saccharomyces cerevisiae (Saccharus omyces cerevisiae), Pichia pastoris and Yarrowia Yarrowia Iipolytica; Baculovirus / insect cell culture (Sum mers et al., A Manual of Methock for Baculovirus Vectors and Insect Cell  Culture Procedures, Texas Agricultukal Experiment Station (1987)); Celloids; and plants (J. Vandekerckhove et al., EIO / TECHNOLOGY 7: 929-932 (1 989)).   Generally, E. Methods for expression of C5a in E. coli have been described by C5a analog gene expression. Actually applicable. Mandecki, Proc. Natl. Acad. Sci. USA 82: 3543-3547 (1985 ); Mollison et al., Proc. Natl. Acad. Sci. USA 86: 292-296 (1989); And Bautsch et al., Immunobiol. 185: 41-52 (1992). Proper adjustment Sequences, such as promoters (eg T7 polymerase, UV5-D, trp or lac), A suitable plasmid vector containing a ribosome binding site and a transcription termination site, and For example, selection of pKK223-2 is within the level of ordinary skill in the art. E. FIG. Departure in Kori In order to optimize the expression, DNA molecules were modified by Guoy et al., Nucleic Acids Res. 10:70 55-7074 (1982). Synthesized using the codon preferred by E. coli, And the synthesized DNA and DNA sequences for several restriction endonuclease sites. It should be possible to facilitate the characterization of possible mutagenesis of the sequence. This approach is Directly and immediately upstream of the triplet that encodes the first amino acid of the peptide. Direct introduction of C5a analogues by introducing an ATG start codon for protein synthesis Expression is possible. In addition, some proteases present in wild-type cells One of the E. E. coli strain lon has the advantage of increased yield of protein Is given. Fraxke et al., Meth. Enzymol. 162: 653-658 (1988).   In general, synthetic genes encoding C5a analogs are expressed in yeast by the following known methods. Can be expressed. For example, Romenos et al., Yeast 8: 423-488 (1992). Section IV of Goeddel (Ed.), Meth. Enzymol. 185: 231-484 (1990); Davi See dow et al., supra; and U.S. Pat. No. 4,775,622. In yeast To optimize expression, the DNA molecule is engineered with endogenous codons using yeast-preferred codons. Prepared to avoid the Arg-Arg pair, which is the target for the KEX2 protease Should be. As opposed to methionine, as the N-terminus of glutamine Use is made of proteolytic cleavage from a signal sequence, such as the alpha factor signal sequence. Promote disconnection. Any possible glycosylation site, such as C5a of the invention. Various embodiments of receptor antagonists are capable of eliminating asparagine at position 64. Preferred.   Expression of genes encoding C5a analogs of the invention in mammalian cells is known to It can be carried out according to the method. Chapter 16, “Expression of Clored Genes in Ma mmalian Cells, ”in Mariatis et al., supra. The current representative method is Berg et al., BioTechnigues 14 (6): 972-978 (1993). Disclosed. Suitable human cells are commercially available cell lines such as HeLA S3 (ATCC CCL2.2 ) And HEK293 (ATCC CRL1573). Expression in CHO cells is eg Asselbergs et al., Fibrinolysis 7: 1-14 (1993). Suitable ham Star cell lines are CHO-K1 (ATCC CCL61), BHK (ATCC CRL 6281), BHK-21 (ATCC 6281). , CCL10 and CRL8544). A typical monkey cell is CV-1 (ATCC CCL70) , COS-7 (ATCC CRL1650) and VERO cells (ATCC CCL81). Suitable mouse details The cell line is C127 (ATCC 1804). A preferred cell line is Uriaub et al., Proc. Natl . Acad. Sci. DHFR-minus CHO as disclosed in USA 77: 4216-4220 (1980). It is a system. More preferred are serum-independent cell lines. Kurano et al., Bio / Technol See ogy 16: 245-258 (1990). In mammalian hosts, the C5a analog Glycosylated or non-glycosylated forms can be produced.   The transformed E. The C5a analog isolated from E. coli cells contains a C-terminal cystein. Is present in a reduced form, i.e. it is preferably using a convenient one-step method Is regenerated to have a biological activity containing a free thiol group. Applicants have found that reducing agent: oxidizing agent at a molar ratio of at least about 100: 1 to about 500: 1. A modified C5a analog having a reducing pair has a C-terminal cysteine in a reduced form. It was surprisingly discovered that it results in a biologically active C5a analog that does. This ratio Is about 10 to about 50 times higher than the known ratio (10: 1 reduced sulfhydryl compound). The preferred ratio of compound: oxidized sulfhydryl compound is U.S. Pat. No. 4,620,948. No. (Builder et al., Column 17, lines 43-45). This way For example, the transformed E. E. coli cells are sufficient to cause the production of C5a analogs. After culturing under conditions, denaturing agents and lysis were used to generate denatured C5a analogs. Agent, eg 6M guanidine HCl, depending on the case Known techniques, eg sonication, Fren Further destroyed by ch Press or Dyno Mill. Next, denatured C5a analog Cells thus mixed or cells disrupted in this way are reconstituted. Under suitable conditions to produce the biologically active C5a analog produced, the At least about 100: 1 to about 500: 1 redox couple in a reducing agent to oxidizing agent molar weight ratio. Mixed together. Suitable redox couples are cysteine / cystine and reduced groups. Includes lutathione / oxidized glutathione. Those skilled in the art will appreciate that other redox couples It will be appreciated that it can be used. The glutathione redox couple is preferred. Suitable Rigorous conditions include a pH of 6.5-7.5, preferably a pH of 7.4. The mixture is tan Allow to stand at room temperature for a time sufficient to maximize protein yield. The preferred time is , About 0.5 hours to about 4 hours. Therefore, this method uses bacterial cells to Ie the insoluble mass of expressed protein) and then C- Eliminates the need to reduce the thiol group of the terminal cysteine.   In others, C5a analogs are described in U.S. Pat.No. 4,620,948 (Builder et al. ) And can be regenerated according to standard regeneration and purification schemes. Those According to the method of C., the C-terminal cysteine is an adduct such as Cys-Cys or Cys- It will exist in the form of glutathione. Therefore, the adduct is a free thiol group. Should be further reduced to obtain Applicants are responsible for the disclosed C5a analogs. The adduct also exhibits substantially no agonist activity as defined herein. , And thus also as a C5a receptor antagonist included within the scope of the present invention. I found it to work. However, the additional reduction is those standard replay techniques. If a method is used, it is necessary to prepare the preferred embodiment.   Following regeneration, the C5a analog can be purified to the desired degree. representative Purification schemes include ultrafiltration, diafiltration, gel electrophoresis, and chromatography. Matography methods such as ion exchange chromatography, size exclusion chromatography Chromatography, HPLC, reverse phase HPLC, Sephadex treatment, dialysis, affinity chromatography Includes chromatography, etc. Those skilled in the art will appreciate that a combination of purification schemes may be used. You will understand that.   C5a analogs with a C-terminal cysteine residue are dimeric according to standard techniques. Can be oxidized to form To prepare the dimer, The thiol (-SH) group of the C-terminal cysteine of the nomer (analog) is disulfide Oxidized to form a bond. Homodimers and heterodimers are referred to by the term " "Dimer".   The C5a analogs and dimers thereof of the present invention include complement systems and more particularly C5a a and the treatment and / or prognosis of injury conditions or diseases including analphylatoxins. Useful for prevention. They face a high risk of C5a-mediated tissue destruction and death. It is most therapeutically effective when administered to any mammalian patient, especially humans. is there. Generally, the condition or disease is that C5a is proteolytic in serum. Is an inflammatory disease produced by Typical conditions that respond to C5a analog treatment are pneumonia, Adult respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis, lung inflammation or injury, chronic progressive lung Separation-cystic fibrosis, cotton lung disease, asbestosis-induced inflammation, myocardial infarction, post-myocardial infarction Inflammation, ischemic heart injury, cirrhosis, primary biliary cirrhosis inflammation, chronic hepatitis, pancreatitis, outbreak Cranial nerves in bloody pancreatitis, inflammatory bowel disease, colitis, ischemic brain injury, encephalitis, meningitis Injury, meningitis, uveitis, Purtscher's retinopathy, immune complex-mediated glomerulonephritis , Renal cortical necrosis, gout, vasculitis, serum sickness, angioedema, myasthenia gravis, generalized erythema Thematosus, rheumatoid arthritis, blisters Skin disorders, hypersensitivity, psoriasis, endotoxic shock, sepsis, severe trauma and burns Including. They can also be used in patients with transplant rejection, immunosuppressive therapy or poisonous infusions. Patients receiving blood, exposed to medical devices and following hemodialysis and leukapheresis It can also be used therapeutically to treat patients experiencing pulmonary insufficiency.   The analogs and dimers thereof are associated with reperfusion, eg ischemia followed by reperfusion, and medical treatment. As a preventive agent and in conditions caused by circulatory contact with drug devices It has additional therapeutic uses to prevent fertility rejection. In this case, the C5a analog is Prior to events known to cause or exacerbate existing inflammatory conditions Is suitably administered at substantially the same time.   The C5a analogs and dimers thereof of the present invention are conventional, non-toxic, pharmaceutically acceptable chimerics. As a dosage unit formulation containing a carrier, an adjuvant and optionally a vehicle. Any therapeutically useful route for proteinaceous medicine, eg parenteral, nasal It can be administered internally, rectally or buccally. The term "parenteral" means subcutaneous, intravenous, muscle It includes delivery systems such as internal, intrasternal, intraarterial injection and infusion techniques.   Dosages of the C5a analogs (and dimers) of the invention may be desired for a particular patient. Can be varied to achieve the desired therapeutic response. This is, for example, a particular antagon Depending on the activity of the strike, the mode of administration, the severity of the condition being treated and the medical condition of the patient. I do. Determination of a therapeutically effective dose for a given condition and patient is within the skill of the art. Within the bell. Generally, about 1 μg to 100 mg / kg body weight per day Dose levels are administered daily to the mammalian host. A preferred dosage level is from about 0.1 mg It is in the range of about 20 mg / kg body weight-day. The C5a analog is one continuous sequence over a long period of time. To the patient. However, the total effective dose can be More than once per day The dose may be divided into, for example, 2-4 divided doses.   The C5a analogs (and dimers) of the present invention include known pharmaceutically acceptable ingredients and It may be incorporated into the composition using both methods of preparation. For example, Remington et al.,Pharmaceutical Sciences, 15th Ed., MacK Pub. (Easton, PA) (1975) Teru. Suitable compositions for parenteral administration include sterile pharmaceutically acceptable aqueous or aqueous solutions. Exist in non-aqueous solutions, dispersions, suspensions or emulsions and are ready for use Present in sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable Representative examples of harsh aqueous and non-aqueous carriers, diluents or vehicles are water, ethanol. Nols, polyols such as glycerol, propylene glycol, polyethylene Lenglycol and suitable mixtures thereof, vegetable oils such as olive oil and pouring Injectable organic esters such as ethyl oleate are included. The flow rate is Use of steps, eg coating material, eg lecithin, maintenance of required particle size (minutes And in the case of sprinkling) and surfactants.   The composition may also include adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents, anti-precipitating agents. Fungal and antifungal agents such as paraben, chlorobutanolphenol and sorbin Acids, isotonic agents such as sugars, sodium chloride, or agents that delay absorption such as Luminium monostearate and gelatin can be included. C5a receptor Anne Tagonists are slow- or sustained-release or targeted administration systems, such as polys. It can be incorporated into Marmatrix, liposomes and microspheres.   Injectable formulations can be filtered by a variety of means, including bacterial retention filters. May be dispersed or dispersed in sterile water, or some other sterile injectable medium immediately before or at the time of use. Can be sterilized by also incorporating a sterilant in the form of a sterile solid composition which may be .   In addition to the C5a analog and any other active ingredient, the suspension may be a suspending agent. , Eg ethoxylated isostearyl alcohol, polyoxyethylene Sorbitol and sorbitan esters, microcrystalline cellulose, aluminum meta Contains hydroxide, bentonite, agar, tragacanth, and mixtures thereof. Can be.   Compositions for rectal or vaginal administration will usually be in a suitable nonirritating excipient or carrier. -For example cocoa butter, polyethylene glycol or solid at room temperature, However, it is liquid at body temperature and therefore melts in the rectum or vagina and Mixing the polypeptides of the invention with a suppository wax that releases the antagonist. Present in the form of suppositories which may be prepared by   Ophthalmic formulation, eye ointments, powders and solutions are also included within the scope of this invention.   Polyclonal and monospecific specific for the C5a analogs and dimers of the invention. Ronal antibodies can be prepared according to standard techniques. The polyclonal antibody is C5a The analog-carrier protein conjugate is applied to animals such as rabbits, goats, sheep or horses. And the production of anti-C5a analog antibodies. Was For example, A. Johnstone and R. Thorpe,Immunochemistry in Proctice,Blackwe ll Scientific Publicatioms, Oxford (1982). C5a analog of the invention Body-specific monoclonal antibodies are available from Kohler and Milstein, Nature256 : 495-97 (1975). Also, Peters, J. et al. H ., (Eds.)Monuclonal Antiboclies， Springer Verlag Berlin ， Heidelberg ， G See also ermany (1992). Polyclonal and specific for C5a analogs Monoclonal antibodies also show substantially no cross-reactivity with human C5a. the term “Substantially no cross-reactivity” refers to the C5a analog of the invention in a biological sample. Endogenously generated in the assay for the body Anti-C5a analog antibodies to human C5a so that no interference with C5a could be detected. It means a very low (negligible) cross-reactivity of.   The C5a analog-specific antibody of the invention is directed to a subject previously antibodyd by the C5a analog. To detect and quantify its circulating C5a analogs, and It is particularly useful for modifying, eg, neutralizing, the activity of a C5a analog that does. Circulation C5a analogs that are capable of being detected according to standard immunological techniques utilizing antibodies. Generally, a fluid or tissue sample is obtained from the subject, and then a C5a analog and Under suitable conditions to allow the detectable detection of immune complexes with the antibody, It is reacted with an antibody specific for the C5a analog administered to the subject. That's it The formation of such immune complexes is an indication of the presence of the analog in the sample. That's it The use of plasma or serum samples for such assays is preferred. However, the organization , Eg certain blood cells, eg “PMNL may also be used. And / or degree can be determined by various methods known in the art, such as radioimmuno Immunoassay, enzyme immunoassay, fluorescent immunoassay, fluorescent microscope, and the like. Can be determined by A suitable qualitative and quantitative immunoassay method is J. Butler,Immunochemistry of Solid-Phase Immunoassay,CRC Press (1991 ).   Circulating C5a analog detection assays typically monitor analog levels during processing. Used to nitrate. Furthermore, the antibodies of the present invention show activity of circulating C5a analogs. Can be conveniently used in pharmaceutical compositions to denature or neutralize. used The amount of antibody will be a molar equivalent of the amount of analog administered. The composition is parenteral to the subject Can be administered. Intravenous administration is especially preferred under emergency circumstances. Antibodies are pharmaceutically acceptable A unit that can be injected with an acceptable vehicle, such as saline or Ringer's solution It will be formulated in a dosage form.   The invention will be further described by the following detailed examples. The examples are It is for the sake of illustration only and is not limiting unless otherwise stated. Yes.Example 1 Synthesis of the gene encoding human C5a   The human C5a gene was synthesized by oligonucleotide coupling. This case The codon usage of the adult gene is described in E. Designed for optimal expression in E. coli. This synthetic method is shown in FIG. It has an AUG codon more than any other codon Also changed the Thr-to-Met N-terminus because it also confers a higher frequency of translation initiation. It requires the condensation of 5 fragments with residues. Fragment 1 is Shin It encodes the e-Delgarno sequence and the ATG start codon of the synthetic gene. Fragment 2 ~ 5 is C5aOligonucleotide synthesisCode: the oligonucleotide It was synthesized on the Gene Assombler (Pharmacia) by the phase phosphoramidite method. Enough The synthesized oligonucleotide was cleaved from the solid support and concentrated NH_Four5 with OH The protection was released by incubating at 5 ° C for 16 hours. Next, oligonucleo The tide was purified by preparative gel chromatography. Used acrylic The concentration is 10% to 40 bases or less for oligonucleotides of 70 bases or more. Up to 20% for the oligonucleotides. Following electrophoresis, oligonuclear Visualization of cleotide by UV shadowing method, and the main high molecular weight fragmen The gel was cut from the gel. Powder the gel slice in a test tube with a glass rod. Decontamination and 3.0 ml of 0.1 M triethylammonium hydrogen carbonate (TEAB) buffer Incubate at 37 ° C for 16 hours at pH 7.5 The DNA was then extracted.   The rest of the gel was removed by centrifugation and the oligonucleotide was separated by SepPak C- Isolated by chromatography on 18 columns (Waters Associates). The column was loaded with 10 ml of acetonitrile, 5 ml of 30% acetonitrile in 50 mM TEAB and It was pre-equilibrated by successive washes with 10 ml of 25 mM TEAB. Orient Apply gogonucleotides, wash with 10 ml of 25 mM TEAB, and add 35.5 mM TEAB The column was eluted with 5 ml of 50% acetonitrile in the medium. Collect fractions, and 26 Fractions containing the oligonucleotide, as determined by absorbance at 0 nm, were separated by Spee Dried in d Vac (Savant).Oligonucleotide annealing and Coupling : Before annealing, individual oligonucleotides are Oxidation. The kinase reaction mixture is 12 mM MgCl 2.₂, 1 mM DTT (dithiothreate And 77 mM Tris (pH 7.5) in 40 μl total volume containing 2 mM ATP. It contained 1 μg of cleotide. The reaction was loaded with 10 units of T4 polynucleotide kinase. Disclosed and allowed to proceed for 40 minutes at 37 ° C. Add 10 μl of sterile water to each well. Add to the amination reaction and add 48 μl of complementary oligonucleotide to mix. And placed in a heating block at 78 ° C. Turn off the heating block, The mixture was cooled to 30 ° C. The sample is then heated at 68 ° C for 10 minutes in a second heating block , The block was stopped again and the mixture was cooled to 26 ° C. Ani The purified gene fraction was used to transform phage M13mp18 (New England Biolabs). And assembled the gene. In M13mp18, the gene encoding C5a was cloned into The means to assemble required three ligation reactions.   Following individual ligation reactions of the appropriate gene fragments into M13mp18, E. Ko ReJM101 was transformed with the ligated M13 DNA . After isolation of M13 phage from the recombinant clones, sequence analysis of its composition was performed. Was. The final C5a gene was cloned into M13mp18 to obtain M13mp18 / C5a (1-74) Was. C5a (1-74) gene followed by pB from plasmids pTZ19R and pKK223-2 Subcloning into -6 vector to obtain pB-6 / C5a (1-74), where both of the above The plasmid is from Pharmacia. See FIG.Example 2 Site-directed mutagenesis of the C5a gene   Oligonucleotides directed in vitro Mutagenesis System Version 2 (Am ersham), single-stranded DNA from C5a-containing vector M13mp18 / C5a (1-74) and suddenly Site-directed mutagenesis was performed using mutagenic oligonucleotides. C5 The mutagenic oligonucleotide ACGGTGCTTCTGTT with the mutation Cys27Ser in a. Performed according to the method provided by the manufacturer using AACA (SEQ ID NO: 6) . Sequencing of four plaques with dideoxy DNA for correct Cys27Ser mutagenesis Was analyzed by. Double-stranded DNA was isolated from one of the correct mutant clones and Ps Limited by tI and BamHI. A 230 bp fragment containing the mutation is a pB-6 vector Subcloned into The obtained plasmid pB-6 / C5a (1-74, C27S) was , The dideoxy method was used for re-sequencing to confirm the mutation.Example 3 Cassette mutagenesis of the C5a gene   Plasmid pB-6 / C5a (1-74, C27S) or pB-6 / C5a (1-74) was added to EcoRI and And HindIII and in the vector pWCB restricted by the same enzyme. A plasmid pWCB1 that has been cloned and contains a gene encoding C5a (1-74, C27S) A plasmid pWCB100 containing the genes encoding 12 and C5a (1-74) was obtained. next , That plastic Sumid was used for cassette mutagenesis to produce a series of new genes . The oligonucleotides used for cassette mutagenesis should follow the manufacturer's instructions. Applied solid phase phosphoramidite chemistry to the Applied Biosystems 381A DNA Synthesis. manufactured by sizer.   6 μl of a solution containing 60 U of PVUII in 10 μl of TE containing 60 μg of pWCB112 and HindI 3 μl of a solution containing II (New England Biolabs), and 10 × High Salt buffer (1M NaCl, 0.5M Tris / HCl (pH 7.5), 0.1M MgCl₂, 10 mM DTT) 10 μl and 71 μl ddH₂Mixed with O 2. This solution was incubated at 37 ° C for 16 hours. about Preparative electrophoresis of 4.5 kb linearized vector using 1% agarose gel DNA fragment from agarose gel slices that were subsequently purified by The components were electroeluted. Transfer the recovered DNA fragment to an Eppendorf tube, 1 ml absolute ethanol was added and the tube was placed at 14,000 rpm for 10 minutes, Eppendorf. It was centrifuged by a centrifuge. Dry the DNA pellet under vacuum and then Dissolve in 45 μl of TE buffer (10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA) and p Obtain WCB112 / A.   Single-stranded oligonucleotide, 35 bp-sequence, 5'CTGCGTGCTAACATCTCTCACAAAGACATGTGCTA3 '(SEQ ID NO: 7), and 39 bp-sequence 5'AGC Add TTAGCACATGTCTTTGTGAGAGATGTTAGCACGCAG3 '(SEQ ID NO: 8) to 8% polyacrylic acid. Purified by preparative electrophoresis on a mid gel. Following electrophoresis, oligonuc Visualize reotide by UV shadowing and gel appropriate fragments I cut it out from. Grind the gel slice in a test tube with a glass rod, and Incubate the DNA in 3.0 ml of 0.1 M TEAB buffer at pH 7.5 for 16 hours at 37 ° C. It was extracted by filtering. The rest of the gel is removed by centrifugation and Gonucleotides on a SepPak C-18 column Isolated by chromatography on (Waters Associates). Column , 10 ml acetonitrile, 5% 30% acetonitrile in 50 mM TEAB / and 25 mM It was pre-equilibrated by successive washes with 10 ml TEAB. Oligonu Apply cleotide, wash with 10 ml of 25 mM TEAB, and in 35.5 nM TEAB, The column was eluted with 5 ml of 50% acetonitrile.   Fractions were collected and oligonuclear as determined by absorbance at 260 nm. Fractions containing octide were dried in a Speed Vac (Savant). Restricted ve Of 35 and 39 bP to form double-stranded DNA for ligation into the target pWCB112 / A. Rigonucleotide annealing was performed with an equivalent amount of individual oligonucleotides, 0.01 MgCl of M₂ Mix with Klenow buffer containing 0.05M Tris / HCl (pH 7.6) containing Heat the sample to 95 ° C for 10 minutes and cool to room temperature for 2 hours. It was done by Apply double stranded DNA to 1 μl (2U) of T4 DNA ligase (BRL) Thus using the 3-fold excess of insert over the vector, the restricted vector pWCB11 Connected in 2. The reaction was carried out at 4 ° C. for 17 hours.   Many molecules can be labeled with different oligonucleotides and pWs using substantially the same technique. Prepared by simply using either CB100 or pWCB112. They are below It is shown in Table 2.   Analogue numbers 10-20 are agonists and are outside the scope of the present invention. Those Was included for comparison purposes. The preparation of the dimer form of analog no. 8 is described below in 5C. Listed. It is referred to as analog number 22.   The complete nucleotide sequence of the polynucleotide encoding the C5a analog number 21 is It is shown in Table 3 below.   However, one of ordinary skill in the art would appreciate that a large number of polynucleotides encoding the same C5a analog would It will be appreciated that tides can be prepared by the degeneracy of the genetic code. for example For example, Watson et al.,Recombinant DNA, Znd Ed., Freeman, N.M. Y. See (1993) That.Example 4 E. FIG. Expression of C5a and C5a analogs in Escherichia coli   To achieve expression, the synthetic genes for the C5a analogs shown in Table 1 were pWCB. Vector, ie PvuII site changed to deleted BamHI site and PvuI site , And isopropyl-thio-β-D-galactoside (IPTG) -induced ta. Modified expression vector pKK containing the c-promoter and ampicillin resistance gene Subcloned in 223-3 (Pharmacia). E. FIG. Coli strain LCIQ is Goff  et al., Proc. Natl. Acad. Sci. Strain L disclosed in USA 81: 6647-6651 (1984) It is a derivative of C137 (lon, htpR). It is from strain DH5αF'IQ (BRL laboratory) , The F'factor encoding lacIQ, and was the host for the expression plasmid. E. coli containing the appropriate expression plasmid. Coli LCIQ, OD₅₅₀LB buoy until reaches 1 It was grown in Young at 30 ° C. The culture was treated with IPIG at a final concentration of 25 mM. Triggered for 3 hours. Cells are harvested by centrifugation and stored at -80 ° C until use. Was.Example 5a Regeneration and purification of C5a and C5a analogs   Recombinant protein was buffered with 6M guanidinium hydrochloride (5: 1 (v: w), buffer) Solution: cell paste) and thawed with a buffer solution containing frozen E. Stiffness Isolated from an aliquot of vesicle paste. Next, the cells are sonicated to destroy them, To give 1 mM cysteine and 1 mM cystine or 1 mM reduced / acid 50mM Tris / HC containing either modified glutathione to promote regeneration l Dialyzed against buffer (pH 8.0) overnight. The dialysate is then treated with the addition of 6N HCl. Acidified to pH3. The precipitate was removed by centrifugation and the supernatant fluid was removed from Delta Pa Presence of 0.1% TFA on reversed phase HPLC column (Waters) with k C18, 100Å, 15 microns Under water in a 25% to 35% acetonitrile linear gradient over 30 minutes And purified. Collect the major peak eluting from the column at approximately 28% acetonitrile. , And lyophilized. This fraction is a recombinant C5a analog glutathione adak. (Adducts of analogs 5, 7, 8, 9, and 21 in Table 1) or C5a analogs Cysteine adduct (adduct of analog 8 in Table 1) .   C5a analog Gene product cysteine-adduct About 0.002 mmol Was dissolved in 50 ml of 0.1 M Tris buffer (pH 7.4). 0.02 mmol of DTT was added. After 4 hours, about 80% of the C-terminal Cys-Cys bond was converted to free cysteine and The product was loaded onto a C4, 15 micron, 300Å, reverse phase column (Alltech) with 0.1% TFA. Purify for 30 minutes in water using a linear gradient of 25-35% acetonitrile in water. Was. Fractions containing product eluting at about 29% acetonitrile are lyophilized, and Then dried under vacuum and stored at 4 ° C.Example 5b Regeneration and purification of C5a and C5a analogs   Recombinant protein was buffered with 6M guanidinium hydrochloride (5: 1 (v: w), buffer) Solution: frozen cells from Example 4 after thawing with a buffer containing cell paste) E. FIG. Isolated from an aliquot of coli paste. Then the cells are sonicated to destroy , And the product contains 1 mM reduced / 0.01 mM oxidized glutathione  It was diluted 20-fold with 100 mM Tris / HCl buffer (pH 7.4). 4 hours later, the melt The liquor was acidified to pH 3 by the addition of 6N HCl. The resulting precipitate is “centrifuged And 25-35% acetonitrile in the presence of 0.1% TFA. Delta Pak C18, 100Å, 15 micron, reverse phase HPLC column using a linear gradient from Purified on Rum (Waters) for 30 minutes. Color with about 30% acetonitrile The major peak eluting from the membrane was collected and lyophilized. Thus isolated The C5a analog had a C-terminal cysteine with a reduced thiol group.Example 5c Formation of dimers of C5a analogues   C5a analogs with a free thiol group in the C-terminal region were prepared in monomeric form (see Example 5b). (After being reproduced as shown)) .   Recombinant protein was added to 1 mM reduced / 0 in 100 mM Tris / HCl (pH 7.4). Example 4 after regeneration according to Example 5b with .01 mM oxidized glutathione mixture Frozen from E. Isolated from an aliquot of coli paste. After 4 hours, add 6N Acidification to pH 3 after addition of HCl. The precipitate obtained is removed by centrifugation. , And the supernatant was added to SP-Spherodex Equilibrated with 25 mM buffer (pH 7.0). Absorbed on-exchange column. After washing the column with 25 mM Tris (pH 7.0), C Elute the 5a analog from the column with 25 mM Tris (pH 7.0) containing 0.75 M NaCl did. The partially purified C5a analog was brought to pH 3.0 with formic acid and distilled water. Dilute to achieve a protein solution with a conductivity of about 45 mS / cm, and have a N of 0.6 M SP-high performance ion exchange column equilibrated with 50 mM formic acid (pH 3.5) containing aCl Absorbed into. The C5a analog was treated with 0.6-1.0 M NaCl in 50 mM formate buffer (pH 3.5). The column was eluted using a linear gradient.   The major peak eluting from the column at about 0.725M NaCl was collected. Like this The isolated C5a analog is a C-terminal cysteine with a reduced thiol group. It had. Adjusting the pH to 7.0 with 25% aqueous ammonia and storing the solution , Resulted in the conversion of the molecule into its dimeric form. pH 7.0 and approx. 0.3-0.6 mg With a protein concentration of / ml and storage at 4-8 ° C, the conversion rate is completed in 2 days It was at least 80% of the conversion rate. The dimer form of the C5a analog was analyzed for the presence of 0.1% TFA. Delta Pak C18 using a linear gradient of 25% to 40% acetonitrile in situ , 100Å, 15 micron, final on a reversed-phase HPLC column (Waters) for 30 minutes Was purified. Collect the main peak eluting from the column at about 33% acetonitrile, And freeze drying I'm sorry. The molecule thus isolated is isolated from E. Generated by E. coli expression system It was a dimer of a C5a analog.Example 6 Receptor binding assay   C5a and C5a Receptor Antagonists to their Affinity for the C5a Receptor I tested it. Harris et al., J. Receptor Res. 11: 115-128 (1991). Binding of [125I] BH-labeled C5a prepared as described to PMNL membranes. Compatibility was determined by Braunwalder et al., Mol. Immunol. 29 (11): 1319-1324 (1992). With the modifications as listed, Rollins et al., J. Biol. Chem. 263: 520-526 (1988). PMNL to Ca²⁺And Mg²⁺Without hunk Resuspend in 10 mM HEPES (pH 7.3), 2.5 mM MgCl 2.₂， 100 Unit / ml DNAse I, 0.1 mM PMSF, 10 μg / ml aprotonin and 10 μg / ml Of leupeptin. They were then placed in a nitrogen cavitator at 400 psi for 20 minutes at 4 ° C. Equilibration in an ionization cylinder. 25 mM EDTA and the proteins listed above 3 volumes of 0.5M KHCO containing ase inhibitor_ThreeAfter venting inside, gelatinous Material was removed with forceps and the mixture was centrifuged at 400 xg for 10 minutes at 4 ° C. Released. The resulting supernatant was centrifuged at 50,000 xg for 60 minutes at 4 ° C. 200 x 10⁶ Pellets from aliquots showing individual cells were stored at -70 ° C. For binding studies The membranes with 1 mM CaCl 2₂, 5 mM MgCl₂， 0.1mM PMSF ， 0.1% Basitra 20 x 10 in 50 mM HEPES (pH 7.3) containing syn and 0.5% BSA.⁶Re-individual cells / ml Suspended. After an additional 1:75 dilution with the same buffer, add 400 μl of this suspension to 50 μl [125 I] BH-C5a (2200 Ci / mmole specific activity, 4.0 pM final concentration) and C5a analog or 50 μl buffer to be tested at various concentrations for bitter properties To Was added to the containing duplicate tube.   Non-specific binding was determined in the presence of 10 nM unlabeled C5a. The result The combination reaction was initiated by the addition of PMNL membrane and continued at 4 ° C for 120 minutes. Join The released free radioactivity was measured using Cell Haruester (Brandel, Gaithersburg, MD). GF / C glass fiber treated with .05% PEI (polyethyleneimine) for 90 minutes Separated by vacuum filtration through a filter (Whaitman). 3x filter Wash with 5 ml ice-cold 5 mM Tris buffer, pH 7.4, and add multiple wells of Gam It counted by the ma counter (Genesys). Data, non-linear regression analysis program , RS / 1 (Bolt, Beranek and Newman, Bostan), and IC₅₀value Expressed as The results are shown in Table 4 below as Ki values using the Cyeng-Presoff equation. Shown in. See Braunwalder et al., Supra.   The results indicate that the C5a analogs of the invention compete with wild-type C5a with nmoles of Ki. It means that the replacement is done in a static manner.   The C5a analogs of the invention are about 1.0 x 10^-8M or less, preferably about 2.0 × 10^-9M or less And more preferably about 1.0 x 10^-TenBraunwalder et al., Supra, M, supra. Competitive displacement assay (radioligand, [125I] Bolton-Hunter labeled C5 have an affinity for the C5a receptor measured as Ki (in a)) .Example 7 C5a-induced Ca ²⁺ elevation   Recombinant human C5a was dissolved in Hanks buffer containing 0.01% Tween-20, and C5 All stock dilutions of a were made with this buffer. Hula-2 (Hula 2AM, Mole Acetoxymethyl ester of cular Probes) was dissolved in DMSU. Neutrophils end of human From treetop blood to 6% hetastarch (HESPAN, DuPont, WauKegan, IL) Purified by sedimentation in the following, followed by Chapman-Kirkland et al. Immunol. Meth . 142: 95-104 (1991), counter flow elutriation. ). Purified cells (2 x 10⁶/ Ml) with 0.2 μM of Fura-2AM Mix and mix at 37 ° C for 30 minutes without calcium or magnesium without HEPES. Incubated in shocked Hanks solution. 15 minutes before assay, cell suspension Was transferred to a cuvette containing a stir bar and calcium was added to 1 mM. The cell suspension was incubated at 37 ° C with agitation. Assay for 5 hours It was stopped within the cell purification, and a standard control response was periodically obtained, which was the experimental response. Make sure it didn't change over time. The amount of fluorescence is measured by the SLM8000 Determined using a rofluorometer (SLM-Aminco Instruments, Urbana, IL) . Place the cuvette in the fluorometer, and after obtaining a 10 second baseline, remove the antagonist. Add C5a receptor antagonist to be tested for properties and add 340 nm Changes in either fluorescence excitation at / 380 nm (emission at 510 nm) were measured. Addition of analogues After 40 seconds, the challenge dose of C5a was added to a final concentration of 100 pM and the resulting excitation The change in the initiation ratio was measured.   I c₅₀The value was used as a measure of agonist capacity. Their values are 100 pM C5 a C5a analogue required to reduce the calcium elevation response by 50% at a challenging dose Is defined as the concentration of EC₅₀Is the maximum calcium produced by the C5a analog Triggered 50% of the ascending response Defined as the concentration of C5a analog. The results are shown in Table 5 below.   C5a analog No. 7 for 1.0 x 10⁶Tested to a concentration of M. C5a analog No. 8 to 3. 0x10^-6Up to the concentration of M, the analogue No. 9 to 1.5 x 10⁶ Up to the concentration of M and analogue No. 21 to 8.0 x 10^-7Tested to a concentration of M. Agonis No activity was detected in all cases. Analyze with the results shown in Table 2 above The results of the above table show that the analogs of the invention function as competitive inhibitors of C5a. Suggest that. They are uncomplexed cis at the C-terminus of the analog. Thein, or another C5a analog of the invention to achieve the highest potential Show that it should be a cysteine residue that is complexed through a disulfide bond .Example 8 Rabbit skin model of inflammation   All experiments were performed on 2.5-3.0 kg male New Zealand White rabbits. I was broken. The rabbit's back is shaved and 40 to 50 skin areas are marked with different colored mars. Marked by car. Different stimulants (ie C5a, C5a analogues, C5a + C5a  Sterilized, disposable, 26 gauge; analog, vehicle control, etc .; 0.5 inch needle And 1.0 ml using a tuberculin syringe intraperitoneally at 0.1 ml / site. I. D . Injections were administered 45 minutes before euthanasia, repeated 6 times. C5a only has 50ng / site Injected at a dose, and the C5a receptor antagonist was administered at different concentrations at the same dose of C5a. It was co-injected once. Twenty minutes prior to euthanasia, 18-36 μCi [1 25I] -labeled bovine serum albumin is circulated systemically through the atrial appendage vein. Was introduced. At 45 minutes, the rabbits were treated with sodium pentobarbital I.V. V. Excess Was euthanized by. Secured by puncturing the heart with a 5.0 ml sample of peripheral blood , Centrifuge at 2000 rpm for 10 minutes, and collect 1.0 ml plasma and place in plasma. Used as a control to determine the amount of 125 I. After death, the skin on the back is removed , And pinned it to a wooden plate. Blood in the main vasculature of the skin By hand Squeezed out. This method reduces variability between skin sites and reduces background Reduced radioactivity. Next, attach the 15 mm cork punch and a wood punch to the inflammatory lesion. It was punched from the skin by scraping and stored in 12 × 75 nm polystyrene tubes. next The injection site was analyzed for radioactivity content using a Gamma Counter (Genesys). Was. [125I] -Bovine serum albumin is exuded from blood vessels and localized at sites of inflammation The amount of (BSA) was found to be directly proportional to the degree of enhancement in vascular permeability. Was. C5a analog ID₅₀Values generated by 50 ng C5a co-injected at the same site The dose of the C5a analog that causes a 50% reduction in the radioactivity exerted.   C5a analog No. 8 (in Table 1) is 70ng / site ID₅₀Found to have And did not provoke a pro-inflammatory response at a dose of 175 ng / site. this The result is that the analog is an antagonist in vivo and It shows that it does not exhibit the nyst property.Example 9 C5a-induced neutropenia in rabbits   All experiments were performed on 2.5-3.0 kg male New Zealand White rabbits Was. Rabbits combined muscle with 10 mg / kg xylazine and 50 mg / kg ketamine Anesthesia was applied by internal administration. 25 gauge butterfly catheter for infusion use Was inserted into the lateral ear vein. Individual blood samples (0.2 ml) from the central ear artery, 2 Collect a 5 gauge, 5/8 inch needle into a fixed plastic syringe and Then, 7.5% EDTA was added as an anticoagulant. Immediately blood, 10 μl 7.5% EDTA Was squeezed into a centrifuge tube containing. Obtain an early arterial blood sample (# 1) and And then immediately injected intravenously with vehicle or the C5a analog of Example 8 (single injection On). 20 seconds later, the second blood Obtain a sample (# 2) and then at 20 seconds intravenously inject 100 ng C5a in 0.2 ml water. Injected internally (single injection). After 20 seconds, a third blood (# 3) sample was taken. 30 minutes later, second round blood sample (# 4) -20 seconds-C5a injection-20 seconds- -A blood sample (# 5) was performed. Blood sample specific for rabbit blood Automated hematology analysis (Technicon H^*  Evaluated by 1) did. Decreased C5a-induced neutrophil count (number per ml) (blood samples C5a-induced neutrophils determined by comparing # 3 to # 2 and # 5 to # 4 (Cytopenia) between vehicle-treated and C5a analog-treated animals did. C5a analogs do not alter normal to baseline neutrophil counts; ie, C5a analogs It did not show agonistic (C5a-like) properties. Rabbit treated with C5a analog C5a-induced neutropenia in mice compared to vehicle-treated rabbits , 40% and 30 min C5a challenge interval, 67% and 41% respectively, significantly (P> 0.05) blocked. Was harmed. The results show the efficacy of systemic administration of C5a analogs.Example 10   Comparative receptor binding and C5a-induced calci of C5a analogs with the following decapeptides: Elevated Um: H-Ile-Ser-Phe-Lys-Asp-Met-Gln-Leu-Gly-Arg-OH (SEQ ID NO: 52).   Five C5a analogs (numbers 5, 7, 8, 9 and 10 from Table 2) were prepared, and Rely on C5a receptor binding and C5a receptor calcium elevation assay, and Or et al., J. Med. Chem. 35: 402-406 (1992) in Table I (No. 14) Compared to adult decapeptide. The results of this experiment are shown in Table 6 below.   The results show that the tested C5a analogs of the invention are more receptor than decapeptide. 14,000 to 10,000-fold higher binding affinity. The above data In addition, the C5a analogs of the invention show substantially no agonist activity (where Peptide is a C5a receptor antagonist molecule that exhibits significant agonist activity It also shows that.Example 11 Preparation of polyclonal antibody specific for C5a (1-71, C27S, Q71C) antigen preparation Made   1 mg of C5a (1-71, C27S, Q71C) was added to Pierce Chemic according to the manufacturer's instructions. al Co. Using Imject Immunogen EDC Mating Kit from (Rockford, IL, USA) Keyhole Limpet Bonded to 2mg hemocyanin (KLH) I tightened it up. Bonding efficiency is 3,500 cpm¹²⁵I-C5a (New England Nuclear, Boston, M . A.) was added. Final volume of zygote is 2.25 ml, 0 .34 mg C5a (1-71, C27S, Q71C) (0.15 mg / ml) and evaluated 0.9 mg / ml KLH Was included.Generation of anti-C5a (1-71, C27S, Q71C) antiserum   C5a (1-71, C27S, Q71C) conjugate (0.5 ml) was added to 0.5 ml of complete Freund's Ajuba. Homogenization (Sigma Chemical Co., St. Louis, MO). Millbrook Farms (Amherst, MA) female New Zealand White rabbit scapula Two sites (0.2 ml homogenate per site) were injected subcutaneously. 21 days later, above The process was repeated. Additional injections were made with incomplete Freund's adjuvant. The third injection after a total of 55 days and the fourth injection after 126 days. Was. Blood (approximately 30 ml) was taken from rabbits 3-5 after each injection and allowed to clot. And the serum was removed.Peptide immobilization for antibody adsorption   C5a (1-71, C27S, Q71C) or C5a (1 mg) was added to Pierce Chemi as described above. cal Co. From Imject Immunogen EDC Mating Kit from and for subsequent efficiency studies^{12 Five} I-C5a was used to conjugate to 2 mg bovine serum albumin (BSA). C5a (1- 71, C27S, Q71C) / BSA is 2.25 ml at 0.25 mg C5a (1-71, C27S, Q71C); C5a / For BSA, the final volume was 2.25 ml with 0.32 mg / ml C5a. Both joints Contained 0.9 mg / ml of evaluated BSA.   The two peptide conjugates were combined with 0.2M buffered to pH 8.6 with sodium carbonate. It was dialyzed against sodium hydrogen carbonate. For individual conjugates, use the same buffer More prewashed AH (aminohexyl) -agarose gel (Sigma Chemical Co ., St. 2 ml of Louis, MO) was added to 1% (v / V) to a final concentration and incubated at 20 ° C for 15 minutes to activate Sexualized. Wash the gel thoroughly with buffer to remove glutaraldehyde and The conjugate solution was added to and incubated at 20 ° C. for 1 hour. Combined Rinse the missing protein from the gel and leave the remaining binding sites at 0.2 M glycyl With 4 ml of lysine at 4 ℃ Blocked by overnight incubation. Made of 0.5 cm x 10 cm glass gel Dulbecco's phosphoric acid packed in a column and containing 0.1% sodium azide It was thoroughly washed with a buffer solution (pH 7.2) (PBS-A).Affinity chromatography   Sera from rabbits immunized with C5a (1-71, C27S, Q71C) were treated with C5a / BSA. It was passed through the column at 2 ml / hour. The absorbed antiserum emerging from the column was collected. Mosquito Rum was buffered with 0.05M sodium phosphate, brought to pH 7.2 and 0.1 It was washed thoroughly with a 0.5 M NaCl solution containing% sodium azide. Combined The antibody was read at 280 nm and set to have a maximum refraction at 0.20D Detection was performed using an in-line UV monitor. Based on the first two passages of serum, The eluted antibody was collected and immediately dialyzed against PBS-A and then ultrafiltered. More concentrated to about 1 mg / ml. Repeat this step several times for individual serum batches. I returned. Serum will no longer detect proteins eluting from the C5a column , Considered as absorbed.   Next, the absorbed antiserum was treated with C5a (1-71, C27S, Q71C) immunoabsorbent. Passed through the column. Dissolve bound antibody with 3M ammonium thiocyanate Discharged and immediately dialyzed against PBS-A, then concentrated to approximately 1 mg / ml.Example 12a Preparation of labeled antibody for detecting bound C5a (1-71, C27S, Q71C)   Anti-C5a (1-71, C27S, Q71C) antibody eluted from C5a column (ie C5a Antibody which cross-reacts with) is conjugated to alkaline phosphatase: antibody 1. 4 mg of alkaline phosphatase (Thailand VII-T, Sigma Chemical Co.) 5 mg (5,000 units). Glutarde Hydide was added to a final concentration of 0.2% (v / v). Incubate the mixture at 20 ° C for 90 minutes. It was incubated and then dialyzed against PBS-A overnight at 4 ° C. Buffer 1 mM Of 0.05M Tris buffer (pH 8.0) containing magnesium chloride, and at 4 ℃ It was dialyzed overnight.Example 12b Detection of bound C5a (1-71, C27S, Q71C) by ELISA   The specifically purified rabbit anti-C5a (1-71, C27S, Q71C) at 0.57 mg / ml was Diluted 1: 500 with 1M sodium borate / boric acid, pH 8.6. ELISA plate (Maxisorp, Nunc, Naperville, IL) with 100 μl of this solution per well Coated at 0 ° C. for 4 hours. Plates to remove unbound material 3 Washed once. C5a (1-71) diluted appropriately with PBS-A + 1% BSA (PBS / BSA) , C27S, Q71C) or C5a (1-71, C27S, Q71C) standard preparations 100 μl was added to the wells at 4 ° C. for 4 hours. Labeled antibody with 100 μl PBS / Add 1: 3000 in BSA and incubate overnight at 4 ° C. Plate Wash and then the enzyme substrate (for alkaline phosphatase 10% (v / V) p-Nitrophenylphosphine at 1 mg / ml in diethylamine (pH 9.8) (Sigma Chemical Co.) was added. Color development at about 20 ° C in a dark room I proceeded for 5 hours. Plate the Biomek 1000 (Beckman Instruments, CA, U Read at 405 nm using SA).   A standard curve of C5a (1-71, C27S, Q71C) was constructed using the same conditions as above ( Data not shown).Example 12c Specificity of specific affinity-purified anti-C5a (1-71, C27S, Q71C)   Use C5a (1-71, C27S, Q71C) or C5a at 100C for 4 hours at 100 ° l. The microtiter plate was coated with 11 μg / well of the buffer solution. Wash the plate , And the antibody eluted from C5a (1-71, C27S, Q71C) after absorption against C5a Serial dilutions of were made into the wells of the plate with 100 μl PBS / BSA. 20 ° C After 4 hours of incubation in the plate, the plate was washed again. Rabbit antibody binding The mixture was diluted 1: 100 (100 μl / well) in PBS / BSA with goat anti-rabbit / horseradish. It was detected by dish peroxidase (Pierce Chemical Co.). 4 at 20 ° C After incubating with secondary antibody for a period of time, wash the plate and hose Radish peroxidase activity is 2,2'-azinobis (3-ethylbenzo Thiazoline) -6-sulfonic acid diammonium salt ABTS substrate (Pierce Chemical Co. ). After 30 minutes of development, color was read at 405 nm.Example 13 Measurement of C5a (1-71, C27S, Q71C) in rabbit plasma samples   Two anesthetized rabbits (# 1 and # 2) (which in turn are C5a (1-71, C27S , Q71C) was injected intravenously) into a heparin-coated tube. I pulled it. Blood was collected after an additional 30 minutes. Next, C5a (1-71, C27S, Q71C ) Was further injected and blood was recollected after 30 minutes. Repeat this 4 times I returned. The sample is centrifuged to remove blood cells and plasma, and Stored at -20 ° C until used for ELISA.   Samples were diluted in PBS / BSA and ELISA was run against the standard curve shown in Example 11. Was quantified by. Determines the rise of its circulating C5a (1-71, C27S, Q71C) over time did. The activity is C5a (1-71, C27S, Q71C ) Was not detected in samples taken from two rabbits prior to the injection of The specificity of The results also show that the antibody did not show cross-reactivity with rabbit C5a, And C5a (1-71, C27S, Q71C) is not a naturally occurring substance in rabbits Indicates thatExample 14 Standard C5a (1-71, C27S, Q71C) and C5a in rabbit circulation (1-71, C27S, Q71C) Comparison of specific anti-C5a (1-7 as detoxification for C5a (1-71, C27S, Q71C) 1, C27S, Q71C)   Plasma samples obtained from the final time point of rabbit # 2 (from Example 13) were Subjected to a 2-fold dilution and the slope of the resulting curve compared to the slope of the standard curve. The two slopes are parallel, which indicates that C5a (1) was circulating in rabbits. -71, C27S, Q71C) still retains its antigenic properties and the standard C5a (1-71, C 27S, Q71C), and can be recognized in an ELISA in the same manner. As a result Is also an analogue where the C5a (1-71, C27S, Q71C) needs to be removed from the circulation. It also suggests that it is neutralized by this antibody.Example 15 Preparation of monoclonal antibody-C5a (1-71, C27S, Q71C)   Preparation of Monoclonal Antibodies by Kohler and Milstein, Nature256: 495-497 (1 975) and performed in BALB / c mice. C5a The same preparation of (1-71, C27S, Q71C) antigen (bound to KLH) was used to Immunized Immunized with hybridoma system P3 / NSI / 1-Ag4-1 (ATCC TIB 18) Produced by fusing splenocytes from isolated mice Screening cell lines using C5a (1-71, C27S, Q71C) / BSA and C5a / BSA I did it. Similar to the method performed with the polyclonal rabbit antiserum, Reacts with C5a (1-71, C27S, Q71C) However, those antibodies that did not react with C5a were treated with a specific monoclonal antibody. It was used as a body-C5a (1-71, C27S, Q71C) antibody. Antibodies that recognize both Used as a delivery antibody and labeled with alkaline phosphatase.   All publications and patent applications referred to herein are subject to the level of skill of one of ordinary skill in the art. Is displayed. All such publications and patent applications are subject to their respective individual publications. And indicates that the patent application is specifically and individually incorporated by reference. To the same extent as if incorporated by reference.   Various modifications described herein will be apparent to those of skill in the art. Like that Such modifications are within the scope of the invention.

[Procedure of Amendment] Article 184-8 of the Patent Act [Submission date] November 9, 1995 [Correction contents]                          The scope of the claims   1. A C5a receptor antagonist that exhibits virtually no agonist activity, Polypeptide analogue of C5a.   2. An analogue is a C-terminal region which differs from its corresponding C-terminal region of human C5a. Comprising at least two cysteine residues and having at least two amino acids The human C5a analog according to claim 1, which is truncated at its C-terminus by a residue.   3. The human C5a analogue according to claim 1, having a cysteine residue at the C-terminus. body.   4. The human C5a analogue according to claim 2, wherein the cysteine residue is present in the form of an adduct. body.   5. The human C5a analog of claim 2, which is 64 to 72 amino acids in length.   The human C5a analog of claim 2, which is 6.68 to 72 amino acids in length.   7. The human C5a analog of claim 2, which is 70-72 amino acids in length.   The human C5a analog of claim 2, which is 8.71 amino acids in length.   9. The human C5a analog according to claim 7, which is C5a (1-71, Gln71Cys).   Ten. A C5a receptor antagonist that exhibits substantially no agonist activity. The derivative of the human C5a analog according to claim 1.   11. A C5a receptor antagonist that exhibits substantially no agonist activity. The derivative of the human C5a analog according to claim 2.   12． 11. C5a (1-71, His67Phe, Gln71Cys), according to claim 10. Derivatives.   13. C5a (1-71, Cys27Ser, His67Phe, Gln71Cys), according to claim 10. Derivatives.   14. The derivative according to claim 10, which is C5a (1-71, Cys27Ser, Gln71Cys).   15． Including first and second polypeptide analogs of human C5a or derivatives thereof Of dimers each of which has a substantial agonist activity. A C5a receptor antagonist not shown in Figure 1 and having a C-terminal cysteine residue And the cysteine residues of its first and second analogs are linked via a disulfide bond. Are linked together and, in addition, their first and second C5a analogs are the same. Or a dimer which may be different.   16. The C-terminal region of each of the first and second analogs corresponds to its corresponding portion of human C5a. Different by being cleaved by the C-terminal region and at least two amino acid residues The dimer according to claim 15, which is   17． C5a (1-71, Cys27Ser, Gln71Cys) of each of the first and second analogues The dimer according to claim 15.   18． A DNA molecule encoding the human C5a analog according to claim 1.   19. A DNA molecule encoding the human C5a analog according to claim 6.   20. A DNA molecule encoding the human C5a analog according to claim 8.   twenty one. A DNA molecule encoding the human C5a analog according to claim 9.   twenty two. A DNA molecule encoding the human C5a analog according to claim 10.   twenty three. A DNA molecule encoding the human C5a analog according to claim 13.   twenty four. A DNA molecule encoding the human C5a analog according to claim 14.   twenty five. The DNA molecule according to claim 18 is operably linked to a predetermined host. Comprising a promoter capable of functioning , Recombinant DNA molecule.   26. Compatible with a given host, comprising a recombinant DNA molecule according to claim 25. Recombinant plasmid.   27. A host compatible and comprising a recombinant DNA molecule according to claim 18. Recombinant vector.   28. A recombinant stably transformed by the recombinant DNA molecule according to claim 18. Host.   29. Selected from the group consisting of bacteria, yeast, fungi, insects, mammals and plant cells 29. The recombinant host of claim 28.   30. E. FIG. 29. The recombinant host of claim 28, which is E. coli.   31. 2. The analog of claim 1, wherein the analog exhibits substantially no cross-reactivity with human C5a. An antibody specific for a human C5a analog.   32. 32. The antibody of claim 31, wherein the antibody is polyclonal.   33. 32. The antibody of claim 31, wherein the antibody is monoclonal.   34. The human C5a according to claim 10, which exhibits substantially no cross-reactivity with human C5a. An antibody specific for a derivative of an analog.   35. 35. The antibody of claim 34, wherein the antibody is polyclonal.   36. The antibody of claim 34, wherein the antibody is monoclonal.   37. The derivative according to claim 14, which shows substantially no cross-reactivity with human C5a. Specific antibody.   38. 38. The antibody of claim 37, wherein the antibody is polyclonal.   39. 38. The antibody of claim 37, wherein the antibody is molyclonal.   40. The C5a analogue according to claim 15, which shows substantially no cross-reactivity with human C5a. An antibody specific for the dimer of the body or its derivatives.   41. The antibody of claim 17, wherein the antibody exhibits substantially no cross-reactivity with human C5a. An antibody specific for immers.   42. A therapeutically effective amount of the C5a analogue according to claim 1 and a pharmaceutically acceptable carrier. A medicine useful in the treatment of C5a-mediated diseases or inflammatory conditions in mammals comprising the body Medicinal composition.   43. A therapeutically effective amount of a C5a analog derivative according to claim 10 and a pharmaceutically acceptable For the treatment of C5a-mediated diseases or inflammatory conditions in mammals comprising a carrier A pharmaceutical composition for use.   44. A therapeutically effective amount and pharmaceutically acceptable amount of the C5a analog derivative according to claim 14. For the treatment of C5a-mediated diseases or inflammatory conditions in mammals, comprising Useful pharmaceutical compositions.   45. Humans that are C5a receptor antagonists that have virtually no agonist activity A pharmaceutical composition useful in modulating the in vivo activity of a C5a analog, comprising:   The antibody of claim 31, in an amount effective to modulate the activity of the analog, and And a pharmaceutically acceptable carrier.   46. The amount of antibody is effective to substantially neutralize the in vivo activity of the analog. The pharmaceutical composition according to claim 45.   47. A method of treating a C5a-mediated disease or inflammatory condition in a mammal, the method comprising: 42. A method comprising administering the composition of claim 42 to a mammal in need thereof. .   48. A method of treating a C5a-mediated disease or inflammatory condition in a mammal, the method comprising: 43. A method comprising administering the composition according to 43 to a mammal in need thereof. .   49. A method of treating a C5a-mediated disease or inflammatory condition in a mammal, the method comprising: 44. A method comprising administering the composition of claim 44 to a mammal in need thereof. .   50. The composition of claim 42 is supplemented with a supplement sufficient to reduce inflammation. Steps of administration to a mammal at one time for events that cause or aggravate body activation A method for reducing C5a-mediated inflammation in a mammal comprising the steps of:   51. The composition of claim 43 is effective to induce complement activation sufficient to reduce inflammation. Comprising the step of administering to a mammal at one time for an event that causes or exacerbates , Methods for reducing C5a-mediated inflammation in mammals.   52. The composition of claim 44 is used to induce complement activation sufficient to reduce inflammation. Comprising the step of administering to a mammal at one time for an event that causes or exacerbates , Methods for reducing C5a-mediated inflammation in mammals.   53. Humans that are receptor antagonists that have substantially no antagonist activity A method for modulating the activity of a C5a analog in a subject in need thereof. Thus comprising the step of administering the pharmaceutical composition of claim 45 to the subject. Law.   54. Human C5a, a receptor antagonist that has substantially no agonist activity A method for modulating the activity of a receptor in a subject in need thereof 47. A method comprising administering the pharmaceutical composition of claim 46 to the subject.   55. Humans that are C5a receptor antagonists that have virtually no agonist activity A qualitative or quantitative assay for determining a C5a analog in a subject;   Obtain a tissue or fluid sample from the subject, and   Excluding the sample between the antibody of claim 31 and the antibody and its analogs. Contacting under conditions sufficient to allow detectable formation of the epidemiological complex. Where the formation of the immune complex serves as an indicator of the presence of the analog in the subject. Is characterized by Surname.   56. The method of claim 55, further comprising the step of quantifying the analog in the subject. The above assay.   57. C5a receptor antagonists that show substantially no agonist activity A process for producing a physically active C5a analog or a derivative thereof, comprising:   A DN encoding a C5a analog under conditions suitable for causing expression of a DNA molecule. E. coli stably transformed with the A molecule. Culturing E. coli cells;   The cells thus cultured are brought into contact with a denaturing agent and a solubilizing agent to denature them. To produce its C5a analog in a modified form; and   The C5a analog thus modified is combined with at least about 100: reducing agent and oxidizing agent. 1 in a molar ratio of its reducing agent to oxidant in a biologically active Comprising mixing under conditions suitable to produce the C5a analog in various forms .   58. 58. The method of claim 57, wherein the mixing is at a pH of about 6.5 to about 7.5.   59. 58. The method of claim 57, wherein the mixing is for a period of time of about 1/2 to about 4 hours. The manufacturing method described.   60. The redox couple is reduced glutathione / oxidized glutathione, The manufacturing method according to claim 57.   61. E. FIG. The E. coli cells showed that the C5a analog derivative C5a (1-71, Cys27Ser, Gln 71Cys) is stably transformed with a DNA molecule encoding the Manufacturing method.   62. E. FIG. The E. coli cells showed that the C5a analog derivative C5a (1-71, Cys27Ser, His 67Phe, Gln71Cys) is stably transformed with a DNA molecule encoding The manufacturing method described in 57.   63. The reducing agent / oxidizing agent has a molar ratio of at least about 100: 1 to about 500: 1. The manufacturing method according to claim 57.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI A61K 38/00 ACD A61K 39/395 ADM ACJ 9284-4C ADZU ACS 8615-4C C07H 21/04 B 39/395 AAA 9356-4H C07K 14/47 ABJ 9356-4H 16/18 ACV 7804-4B C12N 1/19 ADA 7804-4B 1/21 ADM 9637-4B C12P 21/02 C ADZ 9358-4B 21/08 C07H 21/04 0276 -2J G01N 33/566 C07K 14/47 9281-4B C12N 5/00 B 16/18 9281-4B C C12N 1/19 9051-4C A61K 37/02 ACD 1/21 ABE 5/10 ABC C12P 21/02 ACS 21/08 ACJ G01N 33/566 ABS // (C12N 1/21 AAM C12R 1:19) (C12P 21/02 C12R 1:19) (81) Designated country E (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ), AM, AU, BB, BG, BR, BY, CA, CN, CZ, E E, FI, GE, HU, JP, KG, KP, KR, KZ, LK, LR, LT, LV, MD, MG, MN, NO, NZ, PL, RO, RU, SI, SK, TJ, TT, UA, US , UZ, VN (72) Inventor Galakatos, Nicholas G. United States, New Jersey 07901, Summit, Old Oak Drive 7 (72) Inventor Peppard, Jane Bui. United States, New Jersey 07044, Verona, Forest Avenue 160

Claims (1)

[Claims]   1. A C5a receptor antagonist that exhibits virtually no agonist activity, Polypeptide analogue of C5a.   2. An analogue is a C-terminal region which differs from its corresponding C-terminal region of human C5a. Comprising at least two cysteine residues and having at least two amino acids The human C5a analog according to claim 1, which is truncated at its C-terminus by a residue.   3. The human C5a analogue according to claim 1, having a cysteine residue at the C-terminus. body.   4. The human C5a analogue according to claim 2, wherein the cysteine residue is present in the form of an adduct. body.   5. The human C5a analog of claim 2, which is 64 to 72 amino acids in length.   The human C5a analog of claim 2, which is 6.68 to 72 amino acids in length.   7. The human C5a analog of claim 2, which is 70-72 amino acids in length.   The human C5a analog of claim 2, which is 8.71 amino acids in length.   9. The human C5a analog according to claim 7, which is C5a (1-71, Gln71Cys).   Ten. A C5a receptor antagonist that exhibits substantially no agonist activity. The derivative of the human C5a analog according to claim 1.   11. A C5a receptor antagonist that exhibits substantially no agonist activity. The derivative of the human C5a analog according to claim 2.   12． 11. C5a (1-71, His67Phe, Gln71Cys), according to claim 10. Derivatives.   13. C5a (1-71, Cys27Ser, His67Phe, Gln71Cys), according to claim 10. Derivatives.   14. The derivative according to claim 10, which is C5a (1-71, Cys27Ser, Gln71Cys).   15． Including first and second polypeptide analogs of human C5a or derivatives thereof Of dimers each of which has a substantial agonist activity. A C5a receptor antagonist not shown in Figure 1 and having a C-terminal cysteine residue And the cysteine residues of its first and second analogs are linked via a disulfide bond. Are linked together and, in addition, their first and second C5a analogs are the same. Or a dimer which may be different.   16. The C-terminal region of each of the first and second analogs corresponds to its corresponding portion of human C5a. Different by being cleaved by the C-terminal region and at least two amino acid residues The dimer according to claim 15, which is   17． C5a (1-71, Cys27Ser, Gln71Cys) of each of the first and second analogs The dimer according to claim 15, which is   18． A DNA molecule encoding the human C5a analog according to claim 1.   19. A DNA molecule encoding the human C5a analog according to claim 6.   20. A DNA molecule encoding the human C5a analog according to claim 8.   twenty one. A DNA molecule encoding the human C5a analog according to claim 9.   twenty two. A DNA molecule encoding the human C5a analog according to claim 10.   twenty three. A DNA molecule encoding the human C5a analog according to claim 13.   twenty four. A DNA molecule encoding the human C5a analog according to claim 14.   twenty five. The DNA molecule according to claim 18 is operably linked to a predetermined host. Comprising a promoter capable of functioning , Recombinant DNA molecule.   26. Compatible with a given host, comprising a recombinant DNA molecule according to claim 25. Recombinant plasmid.   27. A host compatible and comprising a recombinant DNA molecule according to claim 18. Recombinant vector.   28. A recombinant stably transformed by the recombinant DNA molecule according to claim 18. Host.   29. Selected from the group consisting of bacteria, yeast, fungi, insects, mammals and plant cells 29. The recombinant host of claim 28.   30. E. FIG. 29. The recombinant host of claim 28, which is E. coli.   31. 2. The analog of claim 1, wherein the analog exhibits substantially no cross-reactivity with human C5a. An antibody specific for a human C5a analog.   32. 32. The antibody of claim 31, wherein the antibody is polyclonal.   33. 32. The antibody of claim 31, wherein the antibody is monoclonal.   34. The human C5a according to claim 10, which exhibits substantially no cross-reactivity with human C5a. An antibody specific for a derivative of an analog.   35. 35. The antibody of claim 34, wherein the antibody is polyclonal.   36. The antibody of claim 34, wherein the antibody is monoclonal.   37. The derivative according to claim 14, which shows substantially no cross-reactivity with human C5a. Specific antibody.   38. 38. The antibody of claim 37, wherein the antibody is polyclonal.   39. 38. The antibody of claim 37, wherein the antibody is molyclonal.   40. The C5a analogue according to claim 15, which shows substantially no cross-reactivity with human C5a. An antibody specific for the dimer of the body or its derivatives.   41. The antibody of claim 17, wherein the antibody exhibits substantially no cross-reactivity with human C5a. An antibody specific for immers.   42. A therapeutically effective amount of the C5a analogue according to claim 1 and a pharmaceutically acceptable carrier. A medicine useful in the treatment of C5a-mediated diseases or inflammatory conditions in mammals comprising the body Medicinal composition.   43. A therapeutically effective amount of a C5a analog derivative according to claim 10 and a pharmaceutically acceptable For the treatment of C5a-mediated diseases or inflammatory conditions in mammals comprising a carrier A pharmaceutical composition for use.   44. A therapeutically effective amount and pharmaceutically acceptable amount of the C5a analog derivative according to claim 14. For the treatment of C5a-mediated diseases or inflammatory conditions in mammals, comprising Useful pharmaceutical compositions.   45. Humans that are C5a receptor antagonists that have virtually no agonist activity A pharmaceutical composition useful in modulating the in vivo activity of a C5a analog, comprising:   The antibody of claim 26 in an amount effective to modulate the activity of the analog, and And a pharmaceutically acceptable carrier.   46. The amount of antibody is effective to substantially neutralize the in vivo activity of the analog. The pharmaceutical composition according to claim 45.   47. A method of treating a C5a-mediated disease or inflammatory condition in a mammal, the method comprising: 42. A method comprising administering the composition of claim 42 to a mammal in need thereof. .   48. A method of treating a C5a-mediated disease or inflammatory condition in a mammal, the method comprising: 43. A method comprising administering the composition according to 43 to a mammal in need thereof. .   49. A method of treating a C5a-mediated disease or inflammatory condition in a mammal, the method comprising: 44. A method comprising administering the composition of claim 44 to a mammal in need thereof. .   50. The composition of claim 42 is supplemented with a supplement sufficient to reduce inflammation. Steps of administration to a mammal at one time for events that cause or aggravate body activation A method for reducing C5a-mediated inflammation in a mammal comprising the steps of:   51. The composition of claim 43 is effective to induce complement activation sufficient to reduce inflammation. Comprising the step of administering to a mammal at one time for an event that causes or exacerbates , Methods for reducing C5a-mediated inflammation in mammals.   52. The composition of claim 44 is used to induce complement activation sufficient to reduce inflammation. Comprising the step of administering to a mammal at one time for an event that causes or exacerbates , Methods for reducing C5a-mediated inflammation in mammals.   53. Humans that are receptor antagonists that have substantially no antagonist activity A method for modulating the activity of a C5a analog in a subject in need thereof. Thus comprising the step of administering the pharmaceutical composition of claim 45 to the subject. Law.   54. Human C5a, a receptor antagonist that has substantially no agonist activity A method for modulating the activity of a receptor in a subject in need thereof 47. A method comprising administering the pharmaceutical composition of claim 46 to the subject.   55. Humans that are C5a receptor antagonists that have virtually no agonist activity A qualitative or quantitative assay for determining a C5a analog in a subject;   Obtain a tissue or fluid sample from the subject, and   Excluding the sample between the antibody of claim 31 and the antibody and its analogs. Contacting under conditions sufficient to allow detectable formation of the epidemiological complex. Where the formation of the immune complex serves as an indicator of the presence of the analog in the subject. Is characterized by Surname.   56. The method of claim 55, further comprising the step of quantifying the analog in the subject. The above assay.   57. C5a receptor antagonists that show substantially no agonist activity A process for producing a physically active C5a analog or a derivative thereof, comprising:   A DN encoding a C5a analog under conditions suitable for causing expression of a DNA molecule. E. coli stably transformed with the A molecule. Culturing E. coli cells;   The cells thus cultured are brought into contact with a denaturing agent and a solubilizing agent to denature them. To produce its C5a analog in a modified form; and   The C5a analog thus modified is combined with at least about 100: reducing agent and oxidizing agent. 1 in a molar ratio of its reducing agent to oxidant in a biologically active Comprising mixing under conditions suitable to produce the C5a analog in various forms .   58. 58. The method of claim 57, wherein the mixing is at a pH of about 6.5 to about 7.5.   59. 58. The method of claim 57, wherein the mixing is for a period of time of about 1/2 to about 4 hours. The manufacturing method described.   60. The redox couple is reduced glutathione / oxidized glutathione, The manufacturing method according to claim 57.   61. E. FIG. The E. coli cells showed that the C5a analog derivative C5a (1-71, Cys27Ser, Gln 71Cys) is stably transformed with a DNA molecule encoding the Manufacturing method.   62. E. FIG. The E. coli cells showed that the C5a analog derivative C5a (1-71, Cys27Ser, His 67Phe, Gln71Cys) is stably transformed with a DNA molecule encoding The manufacturing method described in 57.   63. The reducing agent / oxidizing agent has a molar ratio of at least about 100: 1 to about 500: 1. The manufacturing method according to claim 57.