WO1994004173A1

WO1994004173A1 - Therapeutic compositions and methods for inhibiting il-8-mediated inflammation

Info

Publication number: WO1994004173A1
Application number: PCT/US1993/007919
Authority: WO
Inventors: Bernard M. Babior; Yaacov Matzner
Original assignee: The Scripps Research Institute; Hadasit Medical Research Services And Development Company Ltd.
Priority date: 1992-08-21
Filing date: 1993-08-20
Publication date: 1994-03-03
Also published as: AU5087393A; IL106755A0

Abstract

Therapeutic compositions of an IL-8 inactivating agent having the activity of C5a serine protease and methods for inhibiting IL-8-mediated inflammation in patients are described. Also described are diagnostic assays and kits for the detection of an IL-8 inactivating agent having the required activity.

Description

THERAPEUTIC COMPOSITIONS AND METHODS FOR INHIBITING IL-8-MEDIATED INFLAMMATION

Statement of Government Support

This invention was made with the support of the United States Government, and the United States Government may have certain rights in the invention pursuant to the National Institutes of Health Contracts AI24227.

BACKGROUND OF THE INVENTION

1. Field of the invention

The present invention relates to inactivators and their role in inflammatory reactions. More specifically, the present invention relates to therapeutic methods of using compositions of those inactivators for inhibition of IL-8-mediated inflammation by inactivating IL-8.

2. Description of Related Art

During an inflammatory process, neutrophils are attracted to the site of inflammation by chemotactic factors, released first by the causative agent (e.g., an invading microorganism) and then by the neutrophils themselves. One of the major chemotactic factors produced in the course of an inflammatory reaction is the complement fragment C5a, which is released not only through complement activation but, in addition, through the action of a protease that is secreted from neutrophil specific granules. See, Ward et al., J. Immunol..

104:535-543 (1970) and Wright et al., J. Immunol.. 119:1068-1076 (1977). C5a-mediated chemotaxis is thus a self-amplifying process in which low concentrations of the chemotactic factor stimulate the neutrophils to migrate to sites of inflammation, where higher local C5a concentrations provoke the cells into discharging the C5-splitting enzyme from their specific granules, resulting in the production of more C5a that then attracts and activates more neutrophils. Because of this property of self-amplification, the accidental release of even minimal amounts of C5a in the tissues could easily lead to an inappropriate full scale inflammatory response, unless some countervailing mechanism existed to prevent such an occurrence.

C5a is only one of a number of chemotactic peptides that participate in an inflammatory reaction. See, Oppenheim et al., Annu. Rev. Immuno.. 9:617-648 (1991); Baggiolini et al., J. Clin. Invest. 84:1045-1049 (1989) and

Schroder et al., J. Immunol.. 144:2223-2232 (1990). Most of the other chemotactic peptides comprise a single family of homologous peptides known as "chemokines". The members of this family include platelet factor 4, mononuclear cell-derived chemotaxin, granulocyte-activating mediator, neutrophil-activating peptide 2 (NAP-2) and growth regulating gene (GRO).

The prototype chemokine is a cytokine designated interleukin-8 (IL-8; also known as neutrophil-activating peptide 1 , or NAP-1). See Baggiolini et al., supra: Schroder et al., supra: Peveri et al., J. Exp.. Med.. 167:1547-1559 (1988). IL-8/NAP-1 , an 8 kilodalton (kDa) protein, appears to be identical to monocyte-derived neutrophii chemotactic factor (MDNCF) [Matsushima et al., J. EXP. Med.. 167:1883-1893 (1988) and PCT No. WO 89/10962] and neutrophii activating factor (NAF) [Lindley et al., Proc. Natl. Acad. Sci. USA. 85:9199-9203 (1988); PCT No. WO 89/04836].

As the various names indicate, IL-8 or its N-terminus processed variants are secreted by different types of mononuclear phagocytes at sites of inflammation, in response to lipopolysaccharides (LPS), tumor necrosis factor (TNF), or other cytokines, such as interleukin 1 (IL-1). Cytokines mediate the intricate bidirectional interactions between leukocytes and vascular ceils in producing hemostasis as well as inflammatory and immune reactions. Cytokines effect endothelial ceils by causing them to release chemoattractants that induce chemotaxis and extravasation of polymorphonuclear cells and monocytes. See, Broudy et al., J. Immunol.. 139:464-468 (1987) and Sieff et al., Blood. 72:1316-1323 (1988). IL-8, in particular, acts on neutrophils in several ways including serving as a chemotaxin, stimulating 0₂-production and neutrophii degranulation, and increasing the expression of integrins and the complement receptor CRI on the neutrophii surface. See, Oppenheim et al., Annu. Rev. Immuno.. 9:617-648 (1991); Baggiolini et al., J. Clin. Invest.. 84:1045-1049 (1989) and Schroder et al., J. Immunol.. 144:2223-2232 (1990).

The recruitment and activation of leukocytes by IL-8 severely disrupts the architecture of vessel walls and underlying tissues, causing injury. IL-8 is a putative mediator of tissue injury in septic shock, acute respiratory distress syndrome (ARDS), and, generally, inflammatory disease or conditions associated with increased TNF production. In addition, IL-8 has recently been detected in bronchoalveolar lavage fluid of patients with early ARDS. In the context of the lung and associated lung diseases, IL-8 is secreted by non-inflammatory cells, fibroblasts, and type II epithelial cells, in response to IL-1 and TNF which are produced by alveolar macrophages. See, Kunkel et al., Exp. Lunα Res.. 17:17-23 (1991). The resulting accumulation of IL-8 induces the recruitment of neutrophils to the pulmonary interstitium and/or airspace. IL-8 is of particular interest because it is capabie of maintaining an inflammatory reaction over time, a property perhaps explained by the resistance of IL-8 to proteolysis and other environmental factors (e.g., pH and temperature), which allows it to remain active and attract neutrophils to a site of inflammation for many hours. See, Peveri et al., J. EXP. Med.. 167:1547-1559 (1988) and Collins et al., J. Immunol.. 146:677-684 (1991).

Blockage of chemotaxis and neutrophii activation during inflammatory disease is a major therapeutic goal. Therapeutic intervention at various points of the inflammatory cascade is required to prevent injury to tissues. Induction of IL-1 and TNF production occurs within thirty minutes following exposure to LPS, for example. Antibodies against the active, receptor-interacting site of TNF are effective intervenors in various animal models, for example, a pig model for septic shock. However, anti-TNF antibodies have to be administered very early in the course of an inflammatory response to be effective.

A therapeutic treatment directed against the later-acting inflammatory IL-8 protein would thus be beneficial. This would permit intervention at the secondary response level, subsequent to production of TNF, affording a larger window for diagnosis and treatment. In seeking such a countervailing mechanism, a serine protease in synovial and peritoneal fluids was discovered that neutralizes the pro-inflammatory complement fragment C5a peptide by means of limited proteolysis. See, Matzner et al., Immunol.. 49:131-139 (1983); Matzner et al., J. Lab. Clin. Med.. 103:227-235 (1984) and Ayesh et al., 1 Immunol.. 144:3066-3070 (1990). The activity of this C5a protease, also referred to as an inactivating protein, was found to be greatly reduced in synovial and peritoneal fluids from patients with familial Mediterranean fever (FMF), an inherited disease characterized by recurrent episodes of unprovoked inflammation of the joints and the pleural and peritoneal cavities. See, Matzner et al., Blood. 63:629-633 (1984); Matzner et al., N. Enαl. J. Med.. 311 :287-290

(1984) and Matzner et al., Arch. Intern. Med.. 150:1289-1291 (1990). These findings suggested that the C5a serine protease plays a regulatory role in preventing inappropriate episodes of inflammation in serosal tissues and that its deficiency in FMF may explain the unprovoked attacks of sterile inflammation characteristic of the disease.

Summary of the Invention

It has now been discovered that serosal fluids can also eliminate the chemotactic activity of IL-8 and, thereby, decrease the inflammatory response. In addition, the agent in serosal fluid responsible for IL-8 inactivation has been identified as a serine protease that also inactivates the complement protein

C5a. Thus, the inactivating protein was previously designated as C5a serine protease. The inactivation of IL-8-mediated inflammation by an agent having the activity of C5a serine protease therefore provides a significant advancement for the regulation of inflammation.

One aspect contemplated by this invention is a method for inhibiting

IL-8-mediated inflammation. This method comprises administering a composition containing a therapeutically effective amount of an IL-8 inactivating agent, or functional equivalents thereof, in a pharmaceutically acceptable excipient. In a preferred embodiment, the IL-8 inactivating agent has the activity of C5a serine protease.

A related aspect contemplates a method for detecting the presence of an IL-8 inactivating agent comprising contacting the agent having the activity of C5a protease with a diagnostically effective amount of IL-8 susceptible to inactivation by the agent. In a preferred embodiment, the IL-8 inactivating agent has the activity of C5a serine protease. The preferred means for detection in this aspect of the invention comprises measuring the activity of any IL-8 present in the sample, the measuring of which is selected from the group consisting of: a cell chemotaxis assay; a myeioperoxidase release assay; a spectrophotometric assay; an IL-8 receptor binding assay; and gel electrophoresis. A further preferred embodiment of this aspect is the use of a sample of body fluid comprising blood, serosal fluid, peritoneal fluid, pieural fluid, bronchoalveolar lavage fluid or synovial fluid from a patient with familial Mediterranean fever such that the increase in chemotaxis is indicative of the absence of the IL-8 inactivating agent.

Further contemplated by the present invention is a kit. The system comprises a package containing, in an amount sufficient to perform at least one assay, IL-8 for assaying for the presence of an IL-8 inactivating agent.

Also contemplated by this invention is a therapeutic composition that is suitable for preventing IL-8-mediated inflammation. Such a composition comprises a therapeutically effective amount of an IL-8 inactivating agent, or functional equivalents thereof, having the activity of C5a serine protease in a pharmaceutically acceptable excipient.

In particular, the advantages that attach to this invention is to intervene at a secondary level in the inflammatory cascade, preventing the amplification of neutrophii migration and additional IL-8 production while permitting additional time for diagnosis and treatment.

Still further embodiments and advantages of the invention will become apparent to those skilled in the art upon reading the entire disclosure contained herein. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the inhibition of IL-8-mediated chemotaxis measured in microns [micrometers, μm)] as a function of exposure time from 0 to 10 minutes with peritoneal fluid that contains the C5a serine protease. The inhibition of IL-8-mediated chemotaxis reflects the inactivation of IL-8. The assays and results are described in Example 3A.

Figure 2 illustrates the inhibition of IL-8-mediated chemotaxis measured in microns [micrometers, μm)] as a function of peritoneal fluid concentration. The assays and results are described in Example 3B.

Figure 3 illustrates chemotaxis measured in microns [micrometers, (μm)] as a function of IL-8 concentration in nanograms/miililiter (ng/ml) in the presence and absence of peritoneal fluid. The neutrophii chemotaxis assays were performed as described in Example 3C. For each assay carried out with peritoneal fluid, indicated on the graph by filled circles, a control assay was carried out under the same conditions except that the peritoneal fluid was replaced by an equal volume of buffer, indicated on the graph by the line with open circles.

Figure 4 illustrates SDS-PAGE of IL-8 treated with the purified C5a-inactivating protease or with phosphate-buffered saline (PBS). The treatment of the samples is described in Example 3D. The samples were then subjected to SDS-PAGE under reducing conditions on an 18% polyacrylamide gel and stained with Coomassie Blue. The PBS-treated IL-8 samples are shown in lanes A which border the C5a serine protease-treated IL-8 sample. Detailed Description of the Invention

A. Definition of Terms

Amino Acid Residue: An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkage. All amino acid residues identified herein are in the natural L-configuration. In keeping with standard polypeptide nomenclature, J. Biol. Chem.. 243:3557-59, (1969) and adopted at 37 CFR §1.822 (b) (2), abbreviations for amino acid residues are as shown in the following Table of Correspondence:

It should be noted that all amino acid residue sequences are represented herein by formulae whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a bond to a radical such as H and OH (hydrogen and hydroxyl) at the amino- and carboxy-termini, respectively, or a further sequence of one or more amino acid residues up to a total of about fifty residues in the polypeptide chain.

Antibody: A polypeptide which chemically binds to a haptenic group, i.e., ligand. Antibodies, as used herein, are immunoglobuiin molecules and immunologically active fragments of immunoglobuiin molecules. Such fragments, known in the art as Fab, Fab'; F(ab')₂ and F_v, are included. Typically, antibodies bind ligands that range in size from about 6 to about 34 Angstroms with association constants in the range of about 10⁴ to 10¹⁰ M^"1 and as high as 10¹² M^'1. Antibodies may be polyclonal or monoclonal (MAb). Antibodies can bind a wide range of ligands, including small molecules such as steroids and prostaglandins, biopolymers such as nucleic acids, proteins and polysaccharides, and synthetic polymers such as polypropylene. An "antibody combining site" is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable region that specifically binds (immunoreacts with) antigen. The term "immunoreact" in its various forms is used herein to refer to binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof. An "antigenic determinant' is the actual structural portion of the antigen that is immunologicaily bound by an antibody combining site. The term is also used interchangeably with "epitope".

Liαand: A molecule that contains a structural portion that is bound by specific interaction with a particular receptor molecule.

Polypeptide and Peptide: Polypeptide and peptide are terms used interchangeably herein for a relatively low molecular weight, i.e., about 210 daltons to about 10 kD amino acid residue sequence and designate a linear series of no more than about 50 amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.

Protein: Protein is a term used herein to designate a linear series of greater than 50 amino acid residues connected one to the other as in a polypeptide.

Pharmaceutically acceptable: Refers to molecular entities and compositions that do not normally produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Receptor: A biologically active proteinaceous molecule that specifically binds to (or with) other molecules (ligands). Receptors can be glycosylated.

B. IL-8 Inactivating Aαents

The present invention describes the identification of a novel and specific role for an IL-8 inactivating agent having the activity of C5a serine protease in mediating inflammatory processes. The role is shown to be an inactivating event on the chemoattractant cytokine lnterleukin-8, hereinafter referred to as IL-8, which results in the inhibition of IL-8-mediated neutrophii migration to the site of inflammation. This inactivation culminates in the amelioration of the inflammatory response maintained by neutrophii degranulation following migration. The interaction of an IL-8 inactivating agent with IL-8 is shown to be a unique interaction, different from the known interaction and cleavage of the complement fragment C5a by C5a serine protease.

Insofar as the inactivation of IL-8 described herein involves the interaction of an IL-8 inactivating agent having the activity of C5a serine protease, the mechanism discovered and described herein is distinct from the inhibition of C5a-mediated inflammation by C5a serine protease because of the role played by IL-8 in the present inflammatory response. The IL-8 dependent inflammation pathway described herein and below in Therapeutic Methods is referred to as IL-8-mediated inflammation to emphasize the requirement for IL-8 in the process.

Thus, according to the present invention, an IL-8 inactivating agent is a macromolecule having the activity of C5a serine protease that interacts with a region of IL-8 such that it inhibits the chemotaxic properties of IL-8. Exemplary methods for measuring the inactivation of IL-8 by an IL-8 inactivating agent of this invention are described in Examples 2 and 3. As shown in this invention, the treatment of IL-8 with the IL-8 inactivating agent results in a structural change in IL-8 as identified by sodium docecyl sulfate-poiyacrylamide gel electrophoresis (SDS-PAGE). Whereas normal IL-8 migrates as a monomer and dimer, IL-8 exposed to the IL-8 inactivating agent runs with faster mobility on SDS-PAGE as a monomer indicating that the IL-8 has been structurally altered by the inactivating agent. Structural alterations contemplated by the mode of inactivation include an acyl transfer reaction typical of serine proteases, by limited proteolysis of the substrate, by cleavage at an active site, and the like.

The IL-8 inactivating agent of this invention has the activity of C5a serine protease. This protease and its interaction and inactivation of the complement fragment C5a has been previously described. See, Ayesh et al., J. Immunol.. 144:3066-3070 (1990). An IL-8 inactivating agent of this invention has the characteristics of C5a serine protease that is a 53 kDa heat stable protein the neutralizes the inflammatory activities of C5a. It is defined as a serine protease as it is inhibited by diisopropyl fluorophosphate (DFP) and the serine protease inhibitor phenylmethylsulfonyifluoride (PMSF). As a serine protease, it belongs to a family of serine proteases whose members include the digestive enzymes chymotrypsin, trypsin, eiastase, the clotting factors prothrombin and factor X, along with plasminogen, prourokinase and t-PA. The serine proteases as a class share approximately 40% amino acid residue sequence homology and structural spatial conformation evidenced by X-ray crystallography.

Despite their similarity, the serine proteases exhibit distinct substrate specificities and functional properties. The C5a serine protease, synthesized by fibroblasts and secreted into synovial, peritoneal, pleural, bronchial and the like fluids, has only been shown previously to inactivate the C5a substrate involved in mediating short-lived inflammatory responses. The C5a serine protease exhibits a K,,, of 10^"6 M and a V_max of 33 nmoles C5a/minute/milligram of protease that neutralizes C5a by means of limited proteolysis at the carboxy terminus. The inactivated C5a thus fails to exhibit chemotaxic activity. In contrast, the chemotactic activity of the peptide N-formyl-Methionyl-Leucyl- Phenylalanine was not inactivated by C5a protease.

The C5a serine protease has now been discovered to have a similar inhibitory activity on the substrate IL-8 which, while being involved in separate and long-lived inflammatory responses, does not exhibit structural similarity to C5a. Thus, the inactivation of the IL-8 inflammatory protein inducing inflammatory responses distinct from those induced by C5a as described herein is a novel and unexpected finding.

A subject IL-8 inactivating agent of this invention having the activity of C5a serine protease includes any analog, fragment, or chemical derivative of an active agent as defined herein, so long as the agent is capable of inhibiting the IL-8-mediated chemotaxis of neutrophils and resulting inflammatory response. Therefore, an IL-8 inactivating agent analog can be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use. In this regard, an IL-8 inactivating agent analog of this invention can contain one or more changes in the polypeptide so long as the homoiog retains its function in one or more of the binding and inhibition assays as defined herein. The term "analog" includes any IL-8 inactivating agent having an amino acid residue sequence substantially identical to a sequence of an IL-8 inactivating agent in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such polypeptide displays the requisite binding activity.

"Chemical derivative" refers to a polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyi groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form 0-acyl or 0-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those agents which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine may be substituted for lysine. IL-8 inactivating agents of the present invention also include any agent having one or more additions and/or deletions or residues relative to the sequence of an agent whose sequence is shown herein, so long as the requisite binding activity is maintained.

The term "fragment' refers to any subject IL-8 inactivating agent having an amino acid residue sequence shorter than that of the native protein.

When a fragment defining a portion of an IL-8 inactivating agent of the present invention has a sequence that is not identical to the sequence of a portion of the agent, it is typically because one or more conservative or non-conservative substitutions have been made, usually no more than about 30 number percent, more usually no more than 20 number percent, and preferably no more than 10 number percent of the amino acid residues are substituted. Additional residues may also be added at either terminus for the purpose of providing a "linker" by which the polypeptides of this invention can be conveniently affixed to a label or solid matrix, or carrier. Preferably the linker residues are not similar in structure to an IL-8 inactivating agent.

Labels, solid matrices and carriers that can be used with the IL-8 inactivating agent analogs of this invention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1 to 10 residues. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. In addition, a subject analog can differ, unless otherwise specified, from the natural sequence of the IL-8 inactivating agent by the sequence being modified by terminal-NH₂ acylation, e.g., acetylation, or thioglycoiic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like.

Any IL-8 inactivating agent of the present invention may be used in the form of a pharmaceutically acceptable salt. Suitable acids which are capable of forming salts with the peptides of the present invention include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, suifuric acid, phosphoric acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or the like.

Suitable bases capable of forming salts with the peptides of the present invention include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g. ethanolamine, diethanolamine and the like).

In other preferred embodiments the IL-8 inactivating agent is conjugated with a carrier molecule. Typical carriers include sepharose, sephadex, proteins, polypeptides and the like. An IL-8 inactivating agent may also be conjugated to itself or aggregated in such a way as to produce a large complex which may be advantageous because it has new biologic properties such as longer half-life in circulation or greater activity.

Also contemplated for use in this invention is an IL-8 inactivating agent having increased activity or specificity accomplished by recombinant DNA techniques familiar to one skilled in the art. For example, site-directed mutagenesis of the DNA encoding an IL-8 inactivating agent of this invention followed by expression of the mutagenized protein is one way of achieving an IL-8 inactivating agent with increased activity.

Additional aspects of the therapeutic compositions contemplated in this invention for use in the method of inhibiting the clinical aspects of IL-8-mediated inflammation are described below in the Sections D and G.

C. IL-8 Related Proteins Inactivated by IL-8 Inactivating Agent lnterleukin-8 (IL-8) is a cytokine that belongs to a novel inflammatory cytokine family that is defined by having a molecular weight from 8 to 10 kilodaltons (kDa) and exhibiting from 20 to 45% homology in amino acid residue sequence having proinfiammatory activities. For review, see,

Oppenheim et al., Ann. Rev. Immunol.. (:617-648 (1991), the disclosure of which is hereby incorporated by reference. Based on familial similarities in structure and function, an embodiment contemplated for use in this invention is the inactivation of protein members of the cytokine family by the IL-8 inactivating agent having C5a serine protease activity, as defined in B. above, on IL-8 as described herein and in Example 3. Contemplated cytokine family members susceptible to inactivation by an IL-8 inactivating agent of this invention include platelet factor 4, beta thromboglobulin, IP-10, melanoma growth factor as referred to as GRO, and the like. These cytokines are members of the intercrine alpha subfamily. Members of the intercrine beta subfamily also contemplated as IL-8 inactivating agent substrates include LD-78, ACT-2, RANTES, and macrophage chemoattractant and activating factor

(MCAF) and the like.

D. Methods for Inhibiting the Inflammatory Response

It has been discovered that an IL-8 inactivating agent of the invention having the activity of C5a serine protease has the capacity to interact with IL-8, and thereby inhibit the neutrophii chemotactic properties of IL-8 the result of which is an inhibition of an inflammatory response. In view of the physiological role in inflammation played by IL-8, such inhibition will block IL-8-mediated inflammation by interfering with neutrophii chemotaxis.

Contemplated by this invention is a method for inhibiting IL-8 chemotactic activity, and thereby preventing inflammation, comprising administering a therapeutically effective amount of an IL-8 inactivating agent having the activity of C5a serine protease or functional equivalents thereof, in a pharmaceutically acceptable excipient.

In the examples herein, the IL-8 inactivating agent having the activity of C5a serine protease is used as an exemplary therapeutic reagent for incorporation in a composition for the present method. However, it should be understood that the invention contemplates the use of functional equivalents of an IL-8 inactivating agent and thus is not limited to that specific reagent.

Thus, the present invention provides for a method for inhibiting IL-8-mediated inflammation. The therapeutic compositions on this invention have a number of uses, and may be used in vitro or in vivo. In vitro, the compositions may be used to block IL-8-mediated chemotaxis of neutrophils at inflammatory sites in bodily fluids, cell cultures, organs and the like materials that may contain IL-8. In vivo, the compositions may be used prophylactically or therapeutically for preventing chemotaxis and thereby preventing IL-8-mediated inflammation and ameliorating the disease states associated with IL-8-mediated inflammation.

In a further embodiment, the invention contemplated the method of inhibiting IL-8-mediated inflammation comprising administration to a patient of a therapeutically effective amount of an IL-8 inactivating agent composition of this invention in a pharmaceutically acceptable excipient. The therapeutic method is directed at delivering the therapeutic IL-8 inactivating agent to the inflammatory site in the body to be treated.

Administration of the therapeutic IL-8 inactivating agent-containing compositions is by a unit dose. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

A therapeutically effective amount of homolog can be expressed as an amount sufficient to produce a final concentration of homolog in a patient's blood. That blood concentration can be determined by an in vitro assay for the homolog in a liquid body sample (e.g., blood), such as described herein, or can be calculated based on the patient's body weight and blood voiume as is well known.

The compositions of the invention are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient without adverse side affects, and degree of inhibition of chemotactic activity of IL-8 on the appropriate cell type. Precise amounts of active IL-8 inactivating agent required to be administered to inactivate the chemotactic activity of IL-8 resulting in an inflammatory response depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of 1 nanomolar to 1 micromolar, preferably 6 to 600 nanomolar, and most preferably about 200 nanomolar in concentration, of IL-8 inactivating agent in an adult, less for a child, and depend on the route of administration. In order to maintain the preferred concentration range of IL-8 inactivating agent in the extracellular fluid, the therapeutic composition must be administered at a dosage of 5 micrograms/hour to 5 milligrams/hour.

The concentration of the IL-8 inactivating agent ingredient in a therapeutic composition will vary, depending upon the desired dosage, use, frequency of administration, and the like. The amount used will be a therapeutically effective amount and will depend upon a number of factors, including the route of administration including aerosols, injectables, oral preparations, suppositories, enemas and the like, the formulation of the composition, the number and frequency of treatments and the activity of the formulation employed. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner and are peculiar to each individual. The in vivo inhibition of IL-8-mediated inflammation by an IL-8 inactivating agent of this invention having the activity of C5a serine protease is desirable in a variety of clinical settings, where a patient is suffering from or at risk for IL-8-mediated inflammation.

Thus, the present invention also contemplates a method of inhibiting

IL-8-mediated inflammation in a patient by the inactivation of IL-8 so as to inhibit the IL-8 chemotactic properties of eliciting the migration of inflammatory cells such as neutrophils from the peripheral blood to the sites of inflammation. The inactivation of IL-8 by the IL-8 inactivating agent, culminating in a inhibition of inflammation, would be clinically useful in patients with various types of inflammation, or at risk of inflammation, including but not limited to patients with very recent myocardial infarction (within 40 hours of the acute event) where the inactivation of IL-8 by an IL-8 inactivating agent of this invention would prevent neutrophii accumulation on exposed tissues due to injury to those tissues, patients with autoimmune responses, general inflammatory or localized inflammatory reactions, glomerular nephritis, delayed type hypersensitivity, psoriasis, autoimmune thyroiditis, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, tissue transplants, graft rejection, and reperfusion injury of tissue.

A preferred therapeutic treatment of this invention is the use of an IL-8 inactivating agent in ameliorating various pulmonary disease states where neutrophii movement and activation are involved. This clinical picture is presented in adult respiratory distress syndrome, idiopathic pulmonary fibrosis, late phase asthma, cystic fibrosis and the like. Previous studies have demonstrated that known chemotactic factors including fibrinogen, fibronectin, the activated cleavage products of complement proteins C3 and C5, specifically C5a and C5a des arg, are not the mechanism for inducing the migration of neutrophils into the pulmonary interstitium and/or alveolus of the lungs in these diseases. Thus, other chemotactic factors must be involved.

In the lungs, IL-8 is produced by mononuclear phagocytic cells (monocytes and/or macrophages) and also by the non-inflammatory fibroblasts and Type II epithelial cells and endothelial ceils of the lung. The production of IL-8 by these cells has been shown to be induced by the nonchemotactic cytokines lnterleukin-1 (IL-1) and tumor necrosis factor (TNF). In response to challenge with as little as 20 picograms per milliliter of TNF-alpha, lung fibroblasts synthesize significant levels of IL-8 within 4 hours and reach a maximum expression at 8 hours. The resulting chemotactic activity of IL-8 on neutrophii migration paralleled this time course and continued for an additional 16 hours. Thus, the progressive inflammatory response mediated by IL-8 begins approximately 4 hours after exposure to nonchemotactic factors released at the onset of an injury. The inhibition of IL-8-mediated inflammation by the IL-8 inactivating agent of this invention has particular therapeutic value as IL-8 itself is not susceptible to proteolysis and other environmental factors such as pH and temperature.

Thus, inhibiting the chemotactic activity of IL-8 by an IL-8 inactivating agent of this invention having the activity of C5a protease results in the inhibition of the neutrophii inflammatory cascade. This therapeutic treatment is contemplated for ameliorating the inflammatory events in lung tissues which are not mediated by C5a, but rather by IL-8. For a review of pulmonary inflammatory disease, see, Kunkel et al., EXP. Lung Res.. 17:17-23 (1991). An additional preferred therapeutic treatment of this invention is the use of an IL-8 inactivating agent in alleviating the inflammatory response seen in patients with familial Mediterranean fever (FMF). Also called recurrent polyserositis, FMF is an inherited disease in which recurrent episodes of unprovoked inflammation affect the joints, pleural space, and peritoneal cavities. The episodes last approximately 2 to 4 days and are accompanied by pain and fever. Patients with FMF have been shown to lack a protein normally found in synovial and peritoneal fluid produced by tissue fibroblasts that antagonizes the chemotactic activity of complement fragment C5a. This inhibitor, characterized as a serine protease as described in B. above, is designated C5a serine protease. See, Matzner et al., N. Enαl. J. Med.. 311 :287-290 (1984) and Matzner et al., J. Cell. PhvsioL 129:215-220 (1986). Thus, screening for patients having FMF by methods as described in Examples 2 is contemplated.

In acute inflammation seen in FMF patients and other inflammatory diseases with the exception of the lung, the production of the neutrophii chemoattractants, C5a and IL-8, are sequential in time and thus are independent mediators of inflammation. C5a, which is a short-acting chemotaxin with a half-live of minutes, is first generated from cleavage of the complement protein C5 by proteases released at the site of injury, the result of which is the migration of neutrophils to the endothelium and subsequent emigration. This interaction induces the leakage of more complement into the extravascular space which results in production of more C5a and more neutrophii migration. This short-termed inflammatory reaction lasts approximately 2 hours. ln contrast, the synthesis of stable, long-acting IL-8 having a half-life of hours instead of minutes results in a sustained inflammatory response. The production of IL-8 by mononuclear phagocytic cells as well as noninflammatory cells, such as fibroblasts and endothelial cells, is induced by IL-1 and TNF which are nonchemoattractant molecules produced in response to injury to endothelial cells. The time course of IL-8 production as described herein begins approximately 4 hours after injury. Thus, in an acute inflammatory reaction, an IL-8 inactivating agent of this invention having the activity of C5a protease not only alleviates the immediate inflammatory response generated by C5a, but also inhibits the separate and long-term inflammatory response generated by IL-8. Treatment of inflammatory diseases in sites where both C5a and IL-8 are produced is therefore a contemplated embodiment of this invention.

The therapeutic methods contemplated by this invention are not limited to those clinical spectra discussed herein. Rather, the use of IL-8 inactivating agent having the activity of C5a serine protease is contemplated where it is desirable to inactivate IL-8-mediated inflammation.

The inhibition of IL-8-mediated inflammation can be detected by measuring changes in the amount of neutrophii accumulation at the site of inflammation. For example, the number of neutrophils that accumulate at the site of a sponge placed under the skin can be determined both before and after an IL-8 inactivating agent of this invention is administered to a patient. See, for example, Price et al., J. Immunol.. 139:4174-4177 (1987). E. Diagnostic Methods

The present invention contemplates any method that results in detecting, in a body fluid such as blood, serosal fluid, peritoneal fluid, pleural fluid, bronochoalveolar lavage fluid and synovial fluid, an IL-8 inactivating agent of this invention having the activity of C5a serine protease using a diagnostically effective amount of IL-8.

The method for detecting IL-8 by this means comprises measuring the activity of any IL-8 present after the sample suspected of containing the IL-8 inactivating agent is contacted with IL-8. The measuring of the activity of any IL-8 present in the sample after contacting is preferably selected from the group of methods consisting of a cell chemotaxis assay, a myeloperoxidase release assay, a spectrophotometric assay, an IL-8 receptor binding assay and gel electrophoresis. Those skilled in the art will understand that there are numerous well known clinical diagnostic chemistry procedures that can be utilized to measure any IL-8 present in the admixture. Thus, while exemplary assay methods are described herein, the invention is not so limited.

One method contemplated for detecting the presence of IL-8 inactivating agent in a body fluid comprises the use of a neutrophii cell chemotaxis assay performed as described in Examples 2E and 3A-3D of the specification. The IL-8 inactivating agent having the activity of C5a serine protease in the samples was determined by measuring the chemotaxis of freshly prepared human neutrophils in Boyden chambers by the leading front technique using a mixture of 10% (v/v) sample and purified IL-8 as the chemoattractant as described Zigmond et al., J. Exp. Med.. 137:387-410 (1973), the disclosure of which is hereby incorporated by reference. Isolated neutrophils for use in the chemotaxis assays are placed in the upper chamber of the Boyden apparatus while the target IL-8 is placed in the lower chamber. After a suitable maintenance period, the migration of the number of cells and the distance traversed the filter are determined. An increase in the number is a measure of the chemotactic influence of the target in the lower chamber. The presence of an IL-8 inactivating agent in a body fluid sample is determined by measuring a decrease in migration of the number of cells and the decrease in the migration distance as compared to controls.

In a related IL-8 inactivating agent detection method, the myeloperoxidase release assay is performed as described by Gerard et al. J. Biol. Chem.. 264:1760-1766 (1989) and as described in Example 1 B to measure the release of myeloperoxidase from neutrophils thereby the presence of IL-8 inactivating agent having the activity of C5a serine protease.

Another preferred measuring method for use in this invention to detect the presence of IL-8 inactivating agent is by spectrophotometry assays performed as described by Ayesh et al., J. Immunol.. 144:3066-3070 (1990) , the disclosure of which is incorporated by reference, and as described in Example 1 B. The inactivation of IL-8 can be followed spectrophotometrically, thereby determining the presence of IL-8 inactivating agent in the tested sample.

In a further preferred method for use in this invention to detect the presence of IL-8 inactivating agent is by an IL-8 receptor binding assay performed as described in Example 3E and as described by Ayesh et al., 1 Immunol.. 144:3066-3070 (1990), the disclosure of which is hereby incorporated by reference. Since the activity of the IL-8 inactivating agent having the activity of C5a protease on IL-8 results in the inability of IL-8 to bind to its receptor, the presence of the inactivating agent in a body fluid is determined by measuring a decrease in the binding of the labelled ligand to its receptor. A preferred use of this measuring method is the detection of the presence or absence of the IL-8 inactivating agent in an FMF patient.

An additional measuring method contemplated for use in this invention, is the determination of the effect of the inactivating agent on the structure of IL-8 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a technique familiar to one of ordinary skill in the art and described in Example 1 B. Since the IL-8 inactivating agent-treated IL-8 exhibits a broader and slightly more rapidly migration pattern than untreated IL-8, the detection of this pattern is indicative of the presence of an IL-8 inactivating agent present in a body fluid sample.

To perform diagnostic assays, as described, diagnostic systems in kit form are contemplated.

F. Diagnostic Kits A diagnostic kit of the present invention useful for in vitro detection of the presence of IL-8 inactivating agent in an in vivo sample is contemplated. The system comprises a composition containing IL-8. The diagnostic system comprises a package containing purified IL-8 that is susceptible to inactivation by an IL-8 inactivating agent of this invention.

A diagnostic system of the present invention in kit form includes, in an amount sufficient to perform at least one assay, a composition containing IL-8 as a separately packaged reagent. Instructions for use of the packaged reagent are also typically included. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like. Also included, in one form or another, may be charts, graphs and the like of predetermined concentration levels correlating specific physiological conditions to levels of IL-8.

The detection of the IL-8 inactivating agent in the sample through the use of a kit is accomplished by measuring the activity of any IL-8 in the sample in assays selected from the group consisting of a cell chemotaxis assay, a myeloperoxidase release assay, a spectrophotometric assay, an IL-8 receptor binding assay and gel electrophoresis, the descriptions of which are presented in Examples 1-3. In addition to these methods of measuring IL-8 to determine the presence or absence of an IL-8 inactivating agent of this invention, the products resulting from neutrophii degranulation following IL-8 receptor activation via IL-8 binding can be detected. The presence of the resultant azurophil granules and superoxide production can be detected with methods familiar to one skilled in the art.

Also contemplated in this invention is a diagnostic kit useful for in vitro detection of the presence of IL-8 inactivating agent in an in vivo sample. This alternative kit comprises a composition containing antibody molecules that immunoreact with an IL-8 inactivating agent of this invention, such as C5a serine protease. The antibody molecules are, or will become, linked to an in vitro indicating means, such as an enzyme indicating means. The diagnostic system comprises a package containing antibody molecules that immunoreact with an epitope present an IL-8 inactivating agent. A diagnostic system of the present invention in kit form includes, in an amount sufficient to perform at least one assay, a composition containing polyclonal or monoclonal anti-IL-8 inactivating agent antibody molecules, or fragments thereof, as a separately packaged reagent, together with a label that indicates the presence of an immunoreaction product. Instructions for use of the packaged reagent are also typically included.

"Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/ sample admixtures, temperature, buffer conditions and the like. Also included, in one form or another, may be charts, graphs and the like of predetermined concentration levels correlating specific physiological conditions to levels of IL-8 inactivating agent.

A diagnostic system of the present invention also includes a label or indicating means capable of signaling the formation of a specifically bound complex containing an antibody molecule of the present invention.

Further preferred are kits wherein the antibody molecules are linked to an enzyme indicating means, such as horseradish peroxidase (HRPO).

As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel methods and/or systems.

The linking of labels, i.e., labeling of, polypeptides and proteins is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al., Meth. EnzvmoL 73:3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Avrameas, et al., Scand. J. Immunol.. Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al., Biotech.. 3:889-894 (1984), and U.S. Pat. No. 4,493.795.

The diagnostic systems can also include, preferably as a separate package, a specific binding agent. A "specific binding agent' is a molecular entity capable of selectively binding a reagent species of the present invention, but is not itself an antibody molecule of the present invention. Exemplary specific binding agents are antibody molecules, complement proteins or fragments thereof, protein A and the like. Preferably, the specific binding agent can bind the antibody molecule of this invention when it is present as part of a complex.

In preferred embodiments the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent. In these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "EUSA" format to detect, for example, the presence or quantity of IL-8 inactivating agent by assaying for immunoreactive molecules in a body fluid sample such as serum or plasma. "EUSA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and/or quantify the amount of an antigen or antibody present in a sample. A description of the EUSA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical

Immunology by D.P. Sites et al., published by Lange Medical Publications of Los Altos, CA in 1982 and in U.S. Patents No. 3,654,090; No. 3,850,752; and No. 4,016,043, descriptions of which are all incorporated herein by reference.

Thus, in preferred embodiments, the antibody or antigen reagent component can be affixed to a solid matrix to form a solid support that is separately packaged in the subject diagnostic systems. The reagent is typically affixed to the solid matrix by adsorption from an aqueous medium, although other modes of affixation, well known to those skilled in the art, can be used.

Useful solid matrices are well known in the art. Such materials include the cross-linked dextran available under the trademark SEPHADEXfrom Pharmacia Fine Chemicals (Piscataway, NJ); agarose; polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, IL; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose-or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophiiized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.

The packages discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. Such packages include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic-foil laminated envelopes and the like.

G. Therapeutic Compositions Also contemplated by this invention is a therapeutic composition suitable for inhibiting IL-8-mediated chemotaxis of neutrophils to an inflammatory site and thereby preventing inflammation, comprising an IL-8 inactivating agent having the activity of C5a serine protease or functional equivalents thereof that inactivates IL-8 in a pharmaceutically acceptable excipient. This composition attains its therapeutic effect by combining with and neutralizing the chemotactic properties of IL-8.

In another embodiment, a therapeutic composition is contemplated that is suitable as an analog to IL-8 inactivating agents as described in Section B above having the capacity to function as defined herein for an IL-8 inactivating agent analog, comprising an analog having the activity of C5a protease, or biological equivalents thereof, in a pharmaceutically acceptable excipient. This composition attains its therapeutic effect by recognizing and inactivating the chemotactic active site of IL-8 and thereby exhibits the biological activity of IL-8 inactivating agent as defined herein.

The preparation of therapeutic compositions which contain antibody molecules or polypeptides as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and pH buffering agents which enhance the effectiveness of the active ingredient.

Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with one or more IL-8 inactivating agents of this invention, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the composition is not immunogenic or otherwise able to cause undesirable side effects when administered to a mammal or human patient for therapeutic purposes.

As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified, or formulated into suppositories, ointments, creams, dermal patches, or the like, depending on the desired route of administration. The preparation can also be prepared into aerosols for inhalation, into solution for use in an enema or into an oral preparation in the form of tablets, pills, capsules, sustained release formulations or powders.

The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof, including vegetable oils, propylene glycol, polyethylene glycol and benzyl alcohol (for injection or liquid preparations); and vaseline, vegetable oil, animal fat and polyethylene glycol (for externally applicable preparations). In addition, if desired, the composition can contain wetting or emulsifying agents, isotonic agents, dissolution promoting agents, stabilizers, colorants, antiseptic agents, soothing agents and the like additives (as usual auxiliary additives to pharmaceutical preparations), pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.

Uquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.

A therapeutic composition typically contains an amount of IL-8 inactivating agent of the present invention sufficient to deliver a therapeutically effective amount to the target tissue, typically of at least 0.1 weight percent of active ingredient per weight of total therapeutic composition. A weight percent is a ratio by weight of active ingredient to total composition. Thus, for example, 0.1 weight percent is 0.1 grams of polypeptide per 100 grams of total composition.

Stated differently, a therapeutic composition typically contains from about 1 nanomolar (nM) to about 1 micromolar (μM) of IL-8 inactivating agent as active ingredient, and preferably from 6 about nM to about 600 nM.

A typical dose range for therapeutically administering the IL-8 inactivating agent of this invention, such that it effectively inactivates target IL-8 in blood resulting in a decrease or complete inhibition of IL-8 receptor-expressing neutrophii chemotaxis to an inflammatory site, is determined in the following calculations: Assuming that 10,000 neutrophils are present in a microtiter of blood, that 20,000 IL-8 receptors are present on the surface of neutrophils; that 6 liters of plasma and extracellular fluid are present in an adult human and that the half-life of the IL-8 inactivating agent is 4 hours, it follows that 6 liters of plasma contain 20,000 X 10,000 X 6 X 10⁶ = 1.2 X 10¹⁵ or 2 nanomoles of IL-8 receptors. With a 4 hour half-life of the IL-8 inactivating agent, in order to maintain a steady state concentration of IL-8 inactivating agent which is at least 3 and up to 300 times the concentration of IL-8 receptors in the bloodstream, then the agent must be infused at a rate of approximately 1 nmol/hour which is equivalent to 0.05 mg/hour for an adult. Thus, the estimated dose would be 5 μg/hour up to 5 mg/hr of IL-8 inactivating agent.

This dosage corresponds to a therapeutically effective amount of a polypeptide or fusion protein of this invention such that when administered in a physiologically tolerable composition or contacted with the target IL-8 in biood is sufficient to achieve a concentration of from about 1 nM to about 1 μM, and preferably from about 6.0 nM to about 600 nM.

H. Antibodies

The term "antibody" refers to a receptor molecule produced by B cells that immunoreacts with and binds to an antigen ligand to form an immunoreactant. An antibody is a member of a family of glycosylated proteins called immunoglobuiins, which can specifically combine with an antigen.

The term "antibody" in its various grammatical forms is also used herein to refer to immunologicaliy active portions of immunoglobuiin molecules, i.e., molecules that contain an antibody combining site or paratope.

Exemplary antibody molecules for use in the methods and kits of this invention are intact immunoglobuiin molecules, substantially intact immunoglobuiin molecules and those portions of an immunoglobuiin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab')₂ and F(v).

Fab and F(ab')₂ portions of antibodies are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by methods that are well known. See for example, U.S. Patent No. 4,342,566; and Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, pp118-124 (1083). Fab' antibody portions are also well known and are produced from F(ab')₂ portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred, and is utilized as illustrated herein.

The phrase "monoclonal antibody" (MAb) designates an antibody produced by clones of a single cell called a hybridoma that secretes antibody molecules of a single specificity. The hybridoma cell is formed by fusing an antibody-producing cell and a myeloma or other self-perpetuating cell line. Such antibodies were first described by Kohler and Milstein, Nature. 256:495-497 (1975), which description is incorporated by reference.

"Polyclonal" antibodies (PAb) are antibodies produced by clones derived from different cells that secrete different antibodies that bind to a plurality of epitopes of the immunogenic molecule.

The preparation of antibodies is well known in the art. See, Staudt et al., J. EXP. Med.. 157:687-704 (1983), or Antibodies: A Laboratory Manual. Harlowe and Lane, Eds., Cold Spring Harbor, NY (1988).

1. IL-8 Inactivating Agent Immunogen

The term "immunogen", as used herein, describes an entity that induces antibody production in the host animal. In some instances, the antigen and immunogen are the same entity, while in other instances, the two entities are different.

The word "inoculum" in its various grammatical forms is used herein to describe a composition containing an inactivating agent of this invention, preferably C5a serine protease, as an active ingredient used for the preparation of antibodies against the immunogen. When a protein is used to induce antibodies it is to be understood that it may be used alone, linked to a carrier or as a multimer, but for ease of expression, these alternatives will not always be expressed hereinafter.

For a peptide that contains fewer than about 35 amino acid residues, it is preferable to use the peptide bound to a carrier for the purpose of inducing the production of antibodies.

When coupled to a carrier to form what is known in the art as a carrier-hapten conjugate, an IL-8 inactivating agent of the present invention is capable of inducing antibodies that immunoreact with and neutralize IL-8 . Useful carriers are well known in the art, and are generally proteins themselves.

Exemplary of such carriers are keyhole limpet hemocyanin (KLH), edestin, thyroglobulin, albumins such as bovine serum albumin (BSA) or human serum albumin (HSA), red blood cells such as sheep erythrocytes (SRBC), tetanus toxoid, cholera toxoid as well as poly amino acids such as poly (D-lysine: D-giutamic acid), and the like.

As is also well known in the art, it is often beneficial to bind a protein or peptide to its carrier by means of an intermediate, linking group. As noted above, glutaraldehyde is one such linking group. However, when cysteine is used, the intermediate linking group is preferably an m-maleimidobenxoyl N-hydroxy succinimide (MBS).

Additionally, MBS may be first added to the carrier by an ester-amide interchange reaction. Thereafter, the addition can be followed by addition of a blocked mercapto group such as thiolacetic acid (CH₃COSH) across the maleimido-double bond. After cleavage of the acyl blocking group, a disuifide bond is formed between the unblocked linking group mercaptan and the mercaptan of the added cysteine residue of the synthetic polypeptide.

Other means of immunopotentiation include the use of liposomes and immuno-stimulating complex (ISCOM) particles. The unique versatility of liposomes lies in their size adjustability, surface characteristics, lipid composition and ways in which they can accommodate antigens. In ISCOM particles, the cage-like matrix is composed of Quil A, extracted from the bark of a South American tree. A strong immune response is evoked by antigenic proteins or peptides attached by hydrophobic interaction with the matrix o surface.

The choice of carrier is more dependent upon the ultimate use of the immunogen than upon the determinant portion of the immunogen, and is based upon criteria not particularly involved in the present invention. For example, if an inoculum is to be used in animals, a carrier that does not 5 generate an untoward reaction in the particular animal should be selected.

2. Polyclonal Antibodies

To induce IL-8-neutralizing antibodies, an immunogenic amount of an inoculum of this invention, preferably containing purified C5a serine protease as the active immunogen, is administered, typically by 0 subcutaneous or intramuscular injection, to a mammal such as a mouse, rabbit, goat, horse, human and the like. In a preferred embodiment of this invention, administration was accomplished by multi-site subcutaneous injections into rabbits as described in Example 2B1. The administered (inoculated) mammal is then maintained for a time period sufficient for the polypeptide present as active ingredient in the inoculum to induce production of neutralizing (i.e., inactivating) anti-IL-8 antibodies. If desired, the antibodies elicited can then be harvested, using well-known techniques, and used in preparations for passive immunization (therapeutic administration of neutralizing antibodies) against IL-8 active site, or in diagnostic assays and systems to detect IL-8 in body samples.

The anti-IL-8 inactivating agent antibody so produced is oligoclonal with respect to IL-8, and thus has restricted epitope specificity relative to anti-IL-8 polyclonal antisera. The polyclonal antibody of this invention has the capability of blocking the interaction between the IL-8 inactivating agent of this invention, namely C5a protease, and IL-8. The anti-IL-8 inactivating agent polyclonal antibody so produced can be used in the therapeutic and diagnostic methods and systems of the present invention where it is desired to increase the inflammatory response and to detect the presence of IL-8 inactivating agents present in samples suspected of containing the agent.

Preferably, a polyclonal antibody of this invention is in an immunopurified form. Immunopurified polyclonal antibody compositions are produced by immunoreacting (adsorbing) a polyclonal antisera onto a solid phase containing the polypeptide immunogen used to induce the polyclonal antisera, rinsing the non-or-weakly-immunoreacting antibodies away from the solid phase, and subsequently eiuting and collecting the specifically immunoreacted antibodies to form immunopurified antibody. Immunopurification is a generally well known method in the art, and can be performed under a variety of conditions designed to produce a polyclonal antibody composition having a higher net affinity for the immunopurifying solid phase immunogen than the affinity of the starting polyclonal antisera. Exemplary immunopurification is described in Example 2B.

3. Monoclonal Antibodies

Suitable antibodies in monoclonal form, typically whole antibodies, can be prepared using hybridoma technology described by Niman et al., Proc. Natl. Acad. Sci.. U.S.A.. 80:4949-4953 (1983), the description of which is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with a polypeptide of this invention.

It is preferred that the myeloma cell line be from the same species as the lymphocytes. Typically, a mouse of the strain 129 GIX⁺ is the preferred mammal. Suitable mouse myelomas for use in the present invention include the hypoxanthine-aminopterin-thymidine-sensitive (HAT) cell lines P3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American Type

Culture Collection, Rockville, MD, under the designations CRL 1580 and CRL 1581, respectively.

Spienocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 1500. Fused hybrids are selected by their sensitivity to HAT. Hybridomas secreting the antibody molecules of this invention are identified using the solid-phase radioimmunoassay (RIA) described in Example 4.

A monoclonal antibody composition of the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate polypeptide specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well known techniques.

Media useful for the preparation of these compositions are both well known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

The monoclonal antibody compositions produced by the above method can be used, for example, in diagnostic and therapeutic modalities wherein formation of an IL-8-inactivating agent immunoreaction product is desired. Stated differently, an anti-IL-8 inactivating agent antibody immunoreacts with the IL-8 inactivating agent and preferentially inactivates the IL-8 inactivating agent thereby blocking the interaction between the IL-8 inactivating agent and IL-8.

The anti-IL-8 inactivating agent specific antibody so produced can be used, inter alia, in the therapeutic compositions and methods of the present invention where it is desired to increase the inflammatory response rather than to inhibit it. For example, the anti-IL-8 inactivating agent antibody would be therapeutically beneficial in immunocompromised patients who lack the ability to mount an inflammatory cascade. A therapeutically effective amount of the antibody molecule-containing compositions typically contain about 0.1 milligram to about 20 milligram of antibody as active ingredient per milliliter (mg/ml) of therapeutic composition, and preferably about 1 mg/ml to about 10 mg/ml. A therapeutically effective amount of an antibody of this invention is typically an amount of antibody such that when administered in a physiologically tolerable composition or contacted with a target IL-8 inactivating agent is sufficient to achieve a final concentration of from about 0.1 μg/ml to about 100 μg/ml, preferably from about 1 μg/ml to about 5 μg/ml, and usually about 5 μg/ml.

In addition, the anti-IL-8 inactivating agent specific antibody of this invention is useful in the methods and systems of this invention for detecting the presence of IL-8 inactivating agents present in samples suspected of containing the agent. Specifically, the sample would be treated with a diagnostically effective amount of the preferred antibody capable of blocking the interaction of the agent and IL-8, thereby allowing for the detection of the agent.

Another preferred use of the monoclonal antibody of this invention is in a kit for the detection of the IL-8 inactivating agent from a specimen where the antibody has the preferred activity described herein.

A preferred monoclonal antibody immunoreacts with the IL-8 inactivating agent, namely C5a serine protease.

Also contemplated are monoclonal antibodies having a binding specificity for the same or cross-reacting epitopes, i.e., immunospecific for the same epitope, on the IL-8 inactivating agent as the above preferred anti-IL-8 inactivating agent antibodies, or derived from the above antibodies. Thus, the present invention contemplates a monoclonal antibody, and immunoreactive

fragments thereof, that has the immunospecificity of a monoclonal antibody produced by a hybridoma contemplated by this invention.

Immunoiogical techniques for determining the immunospecificity of a monoclonal antibody are well known in the art, and can include competition binding studies and other cross-reaction assays. See, for example the immunoassays described in Antibodies: A Laboratory Manual. Hariow et al.,

Cold Spring Harbor Laboratory, 1988.

Also contemplated by this invention is the hybridoma cell, and cultures containing a hybridoma cell that produce a monoclonal antibody of this invention.

Examples

The following description provides details of the manner in which particular embodiments of the present invention may be made and used. This description, while exemplary of the present invention, is not to be construed as specifically limiting the invention. Variations and equivalents, now known or later developed, which would be within the understanding and technical competence of one of ordinary skill in this art are to be considered as falling within the scope of this invention.

1. Preparation of Purified Human C5a Serine Protease from Peritoneal Ascites Fluid

A. Preparation of Peritoneal Fluid Samples Containing C5a Serine Protease The protection afforded by the C5a serine protease against the development of an inflammatory reaction seen in normal subjects as compared with patients with familial Mediterranean fever (FMV) as described in Example 2 suggested that this serine protease might have anti-inflammatory actions beyond its ability to inhibit C5a. However, the basis for the specificity was thought to be overall sequence homology of the preferred substrates. In actuality, though, C5a serine protease as shown in this invention, inactivates both C5a and IL-8. While both proteins are approximately 8 kilodaltons (kDa) in size, they are clearly distinct and exhibit only partial homology over a five amino acid residue stretch in the carboxy terminus. That the C5a serine protease would be capable of inactivating the IL-8-mediated chemotaxis process that occurs in an inflammatory response was thus unexpected. The initial experiments demonstrating this effect were performed with peritoneal fluid as the source of the C5a serine protease and recombinant human IL-8 as the target as described in Example 3.

For the characterization of the inhibition of C5a and IL-8 by the C5a serine protease described respectively in Examples 2 and 3 below, peritoneal fluid was obtained from patients with ascites due to alcoholic or post-hepatitic cirrhosis of the liver. After the fluid samples were first cleared by centrifugation at 1500 X g for 10 minutes at 4 degrees Celsius (4° C), they were divided into aiiquots and stored at -70 ^βC. Before use, the cleared fluid was decomplemented by heating at 56 ° C for 30 minutes. B. Preparation of Purified Human C5a Serine Protease from Peritoneal Fluid Samples

Peritoneal fluid from ascites was used as the source of the serine protease for purposes of purification as it was obtainable in liter amounts. The presence of the C5a serine protease in ascites and in ammonium sulfate precipitates was previously determined by the chemotaxis assay as described by Matzner et al., Immunol.. 49:131-138 (1983) and Matzner et al., J. Clin. Lab. Med.. 103:227-235 (1984), since the myeloperoxidase assay was difficult to calibrate in the presence of the yellow, protein rich material. The ascitic fluid was assayed in the chemotactic assay performed as described in Example 2E after heating at 56 ° C as described above. For the purification of C5a serine protease, however, the peritoneal fluid was not decomplemented by heating.

For the purification, ascites was obtained from patients with alcoholic or post-hepatitic cirrhosis of the liver, centrifuged at 1500 x g for 10 minutes at 4° C to remove cells and debris, then divided into 50 milliliter (ml) aliquots that were stored at -20 ^β C until use. The purification procedure was performed at

4° C.

The C5a serine protease was precipitated from 50 ml of peritoneal fluid by bringing the fluid to 35% saturation. After four hours the mixture was centrifuged (15000 x g, 20 minutes) and the supernatant was discarded. The pellet was suspended in 5 ml of 0.01 M Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) (Sigma Chemical Co., St. Louis, MO) at pH 7.4 and desalted over a Sephadex G-10 column (Pharmacia LKB, Piscataway, NJ) (2.4 x 25 cm) equilibrated with the same buffer. Fractions containing the C5a serine protease were then pooled and applied to a DEAE-cellulose column (Whatman

Biosystems, Maidstone, Kent, England) (DE-52, 3 x 6 cm) equilibrated with 0.01 M Tris-HCl at pH 7.4. The active material was eluted with 0.25 M NaCl in the same buffer and collected into 5 ml fractions. The fractions containing the inhibitory activity were pooled, desalted over Sephadex G-10, adjusted to a final buffer concentration of 0.05 M Tris-HCl at pH 7.0 containing 0.1 M KCl and applied to a Blue Sepharose CL-6B column (Sigma Chemical Co.) (1.6 x 10 cm) equilibrated with the same buffer. Elution was performed with the same buffer at a rate of 0.7 ml/minute.

The C5a serine protease eluted with the pass-through while albumin remained on the column. Fractions containing the C5a serine protease were pooled, concentrated to 4 ml by ultrafiltration with a PM-10 membrane (Amicon

Corp., Lexington, MA) and applied to a Sephadex G-100 column (Pharmacia LKB) (1.5 x 48 cm) equilibrated with 0.04 M Tris-HCl at pH 7.4. The column was eluted at a rate of 0.3 ml/minute with the same buffer and collected into 3 ml fractions. The active fractions were subjected to polyacrylamide gel electrophoresis under denaturing conditions and those with the fewest protein bands were pooled. The pooled active fractions were applied to an L-arginine agarose affinity column (Sigma Chemical Co.) (1.2 x 22 cm) that had been equilibrated with 0.04 M Tris-HCl at pH 8.5. The inhibitor activity was eluted at a rate of 0.45 ml/minute with the same buffer containing 0.2 M NaCl and the fractions (3 ml each) with the highest specific activity were pooled and concentrated using a Speedvac concentrator (SVC 100H, Savant). The maximum increase in specific activity was achieved at the end of the purification but before the pure enzyme was concentrated. Activity was rapidly lost from the purified serine protease, so V_max values reported below should be construed as lower limits. C5a-induced myeloperoxidase release from neutrophils was used to locate the C5a serine protease in column fractions collected above. The assay was conducted as described by Gerard et al. J. Biol. Chem..264:1760-1766 (1989), with slight modifications. In detail, rC5a at 1-5 nanomolar (nM) was maintained at 37 ° C for the indicated period (usually 15 minutes) with 0.1 volume of a given fraction. One hundred microliter (μl) portions of the resultant admixture were added to each of 3 wells of a 96-well microtiter plate. Each well then received 25 μl of neutrophils (4 x 10⁶/ml in HBSS/25 mM HEPES [pH 7.4]/0.25% BSA) that had been maintained for 5 minutes at 37 ° C with 5 μg/ml cytochalasin B. Degranulation was allowed to proceed for 5 min at 37 ° C. Myeloperoxidase release was then measured by adding to each well 75 μl of phosphate buffer/o-phenylenediamine-peroxide solution (prepared by admixing 2 volumes 0.1 M NaHP0₄ pH 6.8 with one volume 40 μM o-phenylenediamine containing 0.06% H₂0₂), and maintaining for an additional 5 minute. Finally, the reactions were stopped with 50 μl 3N HCl, and absorbances at 490 nm were recorded using a microtiter plate reader (Pasteur LP2000), determining C5a concentrations from a standard curve constructed using 0 to 10 nM rC5a. The results were corrected for the release of myeloperoxidase in the absence of rC5a.

To determine the specific activity of the C5a serine protease throughout the various stages of purification, spectrophotometry assays were performed. The inactivation of C5a by the C5a serine protease appears to be due to the removal from the C5a molecule of an as yet uncharacterized peptide. See, Ayesh et al., J. Immunol.. 144:3066-3070 (1990). The elimination of this peptide was accompanied by a change in the ultraviolet spectrum of C5a from which the inactivation of C5a can be followed spectrophotometrically. For this purpose, reaction mixtures were prepared containing 3.3 nM C5a, except for

SUBSTITUTE SHEET the assay mixture with the crude peritoneal fluid where 1 nM C5a was used, together with 0.2 ml portions of purified serine protease in 1 ml of 10 mM Tris-HCl (final volume and buffer concentration). The resulting reaction admixtures were maintained at room temperature in a recording spectrophotometer, following the absorbance at 254 nm. Rates were calculated from the differences in absorbance at 1 and 6 minutes. Because these assays were performed using C5a concentrations far below the K„, of the enzyme, the specific activities are expressed as μg C5a/min/μg serine protease/μM C5a.

In a reaction carried out in a cuvette with a 1 cm path length, a loss in absorbance of 0.01 unit corresponded to the inactivation of 0.72 nmoles C5a/ml reaction volume. This loss of absorbance was not observed in experiments containing C5a alone, purified C5a serine protease alone, or a mixture of C5a and the serine protease together with 1 mM of the serine protease inhibitor phenylmethylsulfonylfiuoride (PMSF) (Sigma Chemical Co.). The spectrophotometric results were confirmed by chemotaxis measurements, which showed that no chemotactic activity remained after 15 minutes of incubation.

The purification of C5a serine protease from peritoneal fluid is summarized in Table 1. From 50 ml of ascites fluid, approximately 3 μg of pure C5a serine protease was obtained with the above-described procedure. The purification procedure resulted in a 3000 fold increase in purity.

2. Characterization of Purified C5a Serine Protease

A. Sodium Docecyl Sulfate-Polvacrylamide Gel Electrophoresis ($DS-PAQE)

To determine the molecular weight of the purified C5a serine protease prepared in Example 1 B, SDS-PAGE was performed according to the method of Laemmli [Nature. 227:680-685 (1970], using 12% running gels. The gels were stained with either Coomassie Blue or silver stain. Non-denaturing PAGE was carried out under the same conditions except for the omission of SDS and dithiothreitol. When the purified C5a serine protease was analyzed by SDS-PAGE, a single major band was seen that migrated at M_r 53-56 kDa. A single band was also seen when the purified material was subjected to polyacrylamide gel electrophoresis under non-denaturing conditions.

B. Western Blotting For Western blotting, the purified C5a serine proteases electrophoresed but not stained as described in Example 2A were transferred electrophoretically from the gel to a nitrocellulose membrane. The membrane was blocked to prevent nonspecific immunoreactions by maintaining the blot in PBS containing 5% bovine serum albumin (BSA) for 2 hours at room temperature followed by maintenance with a 1:100 dilution of rabbit antiserum raised against purified C5a serine protease for 1 hour at room temperature to form an immunoreaction product. 1) Preparation of Polyclonal Antibodies Against C5a Serine Protease

To obtain an anti-C5a serine protease antibody, rabbits were immunized with 100-200 μg of purified C5a serine protease prepared in Example 1B emulsified in 1 ml complete Freund's adjuvant with 0.25 ml being injected into each of the four footpads. The animals were boosted 2 weeks later by an identical procedure, then were boosted 3 more times by subcutaneous injections of 100-200 μg C5a serine protease in 1 ml incomplete Freund's adjuvant. Serum was collected o three weeks after the last injection, decomplemented by heating at 56 ° C for 30 minutes, then assayed for activity against the C5a serine protease using the myeloperoxidase and the chemotaxis methods as described respectively in Example 1 B and 2E.

After the maintenance period required for forming immunoreaction 5 products, the membrane was maintained for 1 hour at room temperature with goat anti-rabbit IgG conjugated to alkaline phosphatase (Sigma Chemical Co.) followed by treatment with a solution containing p-nitroblue tetrazolium-HCI (0.33 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.165 mg/ml) to allow for visualization of the immunoreacted products.

0 The antiserum recognized the 53-56 kDa band on a Western blot of partially purified C5a serine protease, while normal rabbit serum recognized nothing. The antiserum did not recognize the protein on a Western blot of unfractionated peritoneal fluid, presumably because the amount was too low to be detected. 2) Preparation of Monoclonal Antibodies Against C5a Serine Protease

Monoclonal antibodies against purified C5a serine protease were also obtained. The purified C5a serine protease prepared in Example 1B was prepared as an immunogen as described above. Balb/c ByJ mice were immunized intraperitoneally (i.p.) with 50 μg of prepared C5a serine protease immunogen in CFA followed by a second and third immunization using the same immunogen, each about three weeks apart, in IFA. The mice received a boost of 50 μg of the prepared immunogen intravenously (i.v.) in normal saline 4 days prior to fusion and a second similar perfusion boost one day later.

The animals so treated were sacrificed and the spleen of each mouse was harvested. A spleen cell suspension was then prepared. Spleen cells were then extracted from the spleen cell suspension by centrifugation for about 10 minutes at 1000 rpm., at 23 ^βC. Following removal of supernatant, the cell pellet was resuspended in 5 ml cold NH₄CI lysing buffer, and was incubated for about 10 minutes.

To the lysed cell suspension were admixed 10 ml Dulbecco's Modified Eagle Medium (DMEM) (GIBCO) and HEPES [4-(2-hydroxyethyl)-1-piperidineethanesulfonic acid] buffer, and that admixture was centrifuged for about 10 minutes at 1000 rpm at 23 ^βC.

The supernatant was decanted, the pellet was resuspended in 15 ml of DMEM and HEPES, and is centrifuged for about 10 minutes at 1000 rpm at 23 °C. The above procedure was repeated. The pellet was then resuspended in 5 ml DMEM and HEPES. An aliquot of the spleen cell suspension was then removed for counting. Fusions were accomplished in the following manner using the non-secreting mouse myeloma cell line P3X63Ag8.653.1, a subclone of line P3x63Ag 8.653 (ATCC 1580). Using a myeloma to spleen ceil ratio of about 1 to 10 or about 1 to 5, a sufficient quantity of myeloma cells were centrifuged into a pellet, washed twice in 15 ml DMEM and HEPES, and centrifuged for 10 minutes at 1000 rpm at 23 ^βC.

Spleen cells and myeloma cells were combined in round bottom 15 ml tubes. The cell mixture was centrifuged for 10 minutes at 1000 rpm at 23^β C, and the supernatant was removed by aspiration. Thereafter, 200 μi of 50 percent (weight per volume) aqueous polyethylene glycol 4000 molecular weight (PEG; ATCC Baltimore, MD) at about 37 ^βC were admixed using a 1 ml pipette with vigorous stirring to disrupt the pellet, and the cells were gently mixed for between 15 and 30 seconds. The cell mixture was centrifuged 4 minutes at 700 rpm.

At about 8 minutes for the time of adding the PEG, 5 ml of DMEM plus HEPES buffer were admixed slowly to the pellet, without disturbing the cells. After 1 minute, the resulting admixture was broken up with a 1 ml pipette, and was incubated for an additional 4 minutes. This mixture was centrifuged for 7 minutes at 1000 rpm. The supernatant was decanted, 5 ml of HT (hypoxanthine/thymidine) medium were slowly admixed to the pellet, and the admixture was maintained undisturbed for 5 minutes. The pellet was then broken into large chunks, and the final cell suspension was placed into T75 flasks (2.5 ml per flask) into which 7.5 ml HT medium were placed previously. The resulting cell suspension was incubated at 37 °C to grow the fused cells. After 24 hours 10 ml of HT medium were admixed to the flasks, followed 6 hours later by admixture of 0.3 ml of 0.04 mM aminopterin. 48 hours after fusion, 10 ml of HAT (hypoxanthine/aminopterin/thymidine) medium were admixed to the flasks.

Three days after fusion, viable cells were plated out in 96-well tissue culture plates at about 2x10⁴ viable cells per well (768 total wells) in HAT buffer medium as described in Kennett et al., Curr. TOP. Microbiol. Immunol.. 81 :77 (1978). The cells were fed seven days after fusion with HAT medium and at approximately 4-5 day intervals thereafter as needed with HT medium. Growth was followed microscopically, and culture supernatants were collected about two weeks later and assayed for the presence of C5a serine protease-specific antibody by solid phase radioimmunoassay (RIA).

Briefly, 50 μl of PBS containing 5 μg/ml of the prepared C5a serine protease immunogen is admixed into the wells of microtiter plates. The plates are maintained overnight (about 16 hours) at 4 ° C to permit the immunogen to adhere to well walls. After washing the wells four times with SPRIA buffer (2.68 mM KCl, 1.47 mM KH₂P0₄, 137 mM NaCl, 8.03 mM Na_jHPO,,, 0.05% Tween-20, 0.1 KlU/ml Traysol, 0.1% BSA, 0.015% NaN₃), 200 μl of SPRIA buffer containing 3% normal goat serum (NGS) and 3% bovine serum albumin (BSA) are admixed to each well to block excess protein binding sites. The plates are maintained for 30 minutes at 20 °C, the wells emptied by shaking, .and blotted dry to form a solid-support, i.e., a solid matrix to which C5a serine protease immunogen is operatively affixed. To each well is then admixed 50 μl of hybridoma tissue culture supernatant to form a solid-liquid phase immunoreaction admixture. The admixture is maintained for 2 hours at 37 °C to permit formation of solid-phase immunoreaction products. After washing the wells as described hereinbefore, 50 μl of ¹²⁵l-labelled goat anti-mouse IgG at (Cappel Laboratories, Downington, PA) 0.25 μg protein per ml are admixed to each well to form a labelling reaction admixture. That admixture is maintained for 1 hour at 37 ^βC to permit formation of ¹²⁵l-labelled solid-phase immunoreaction products. After washing the wells as previously described, the amount of ¹²⁵l-labelled product bound to each well is determined by gamma detection.

Hybridomas were selected from hybridoma cultures that secreted anti-C5a serine protease antibodies into their culture media, and further characterized as described herein.

To produce and purify the resultant C5a serine protease monoclonal antibodies, hybridoma anti-C5a serine protease was cultured in a 5% C0₂, humidified atmosphere at 37 °C in DMEM containing 2 mM L-glutamine, 50 μg per ml gentamycin, 10% fetal bovine serum, 10% horse serum, all from Grand Island Biological Co., Lawrence, MA, 10% NCTC medium from Microbiological Associates, Rockville, MD, 1 mM hypoxanthine and 0.3 mM thymidine, both from Sigma Chemical Co. Cell concentration was kept in the range of about 1-2 X 10⁵ cells per ml of medium to about 1-2 X 10⁶ cells per ml of medium for cell growth, division, and production of antibody. To produce ascites.tumor fluid containing anti-C5a serine protease antibody molecules, 10-week old Balb/c mice are immunologically primed by intraperitoneal injection with 0.3 ml of mineral oil and subsequently intraperitoneally injected with 3-5 X 10⁵ anti-C5a serine protease hybridoma cells. The inoculated mice are then maintained for a time period sufficient for anti-C5a serine protease antibody-containing ascites tumor fluids to accumulate, e.g., for about 10 to about 21 days. The ascites fluid is collected and clarified by centrifugation at 15,000 x g for 1 hour at 4^βC and stored frozen at -20 "C.

Anti-C5a serine protease antibody molecules are isolated from the ascites fluid by subjecting the fluid to fast protein liquid chromatography (FPLC) on a Pharmacia Mono QHR 5/5 anion exchange column in a Pharmacia FPLC System (both from Pharmacia, Inc.) using a 0-0.5 M NaCl gradient in 10 mM Tris, pH 8.0, and following the directions supplied with the column. The anti-Pep-1 antibody molecules so isolated can then be transferred to any physiologically tolerable, diluent desired by dialysis.

Alternatively, anti-C5a serine protease antibody molecules can be isolated from the ascites tumor fluid by precipitation with ammonium sulfate according to the method described by Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, p100-101 (1983). Briefly, that method entails slowly admixing saturated ammonium sulfate to the ascites fluid until about a 45% to about a 50% ammonium sulfate concentration is achieved. The precipitated immunoglobulins are then collected by centrifugation at 2000 x g, preferably 10,000 x g. The precipitate is washed 2 or 3 times in 40% saturated ammonium sulfate. The precipitated anti-C5a serine protease antibody molecules are then dialyzed against 500-1000 volumes of phosphate buffered saline (PBS) or any other physiologically tolerable diluent desired to remove ammonium sulfate. The dialysis fluid is changed several times at intervals of a few hours. The protein concentration of the recovered dialyzed anti-Pep-1 antibody solution is determined by the Lowry method [Lowry et al., J. Biol. Chem.. 193, 265-275 (1951)] using a bovine serum albumin standard.

Confirmation of functional anti-C5a serine protease antibody specificity in either a serum sample (polyclonal) or hybridoma culture supernatant (monoclonal) was determined by Western blot analysis with purified C5a protease as described above and in the various assays as described in Examples 2 and 3.

C. Recovery of the C5a Serine Protease From a Native Gel In order to locate C5a serine protease activity on a native gel, the assumption was made that the serine protease might resemble a streptococcal C5a serine protease that cleaves C5a on the carboxyl side of lysine 68. Therefore, an assay developed to detect proteases that cleave near a basic residue was adapted for use in this invention. This assay, based on the hydrolysis of S-2251, (D-valyl-leucyl-iysine p- nitroanilide 2 HCl) (KabiVitrum, Stockholm, Sweden) a chromogenic substrate that is acted on by such proteases as described by Friberger, Haemostasis. 7:138-145 (1978), was used to locate the C5a inactivating protein in a polyacrylamide gel. S-2251 was found to be hydroiyzed by the purified C5a inactivating protein from peritoneal fluid where the release of p-nitroaniiine was proportional to the quantity of C5a inactivating protein activity and the time of incubation. Two other chromogenic substrates, S-2288 and S-2443, respectively (D-isoleucyl- prolyl-arginine p-nitroanilide 2 HCl) and (L-pyroglutamyl-glycyl-arginine p- nitroanilide 2 HCl) (Kabivitrum) were not hydrolyzed by the C5a inactivating protein. S-2251 was therefore used to locate the C5a inactivating protein on a gel.

A sample of purified C5a serine protease prepared as described in Example 1B was electrophoresed through a 12% polyacrylamide gel

- without SDS or dithiothreitol. After electrophoresis, the gel was cut into

0.5 cm slices each of which was maintained overnight at room temperature with 1 ml of 0.2 mM S-2251 in 40 mM Tris-HCl at pH 7.4.

Cleavage of S-2251 was determined spectrophotomβtrically at 405 nm.

When a neighboring track on the non-denaturing gel was cut into 0.5 cm slices, serine protease activity as measured by the hydrolysis of the artificial substrate S-2251 was found to comigrate with that band. No serine protease activity was detected, however, in a similar experiment in which ovalbumin was electrophoresed on a non-denaturing gel. Thus, the C5a serine protease exhibited substrate specificity.

D. Nonspecific Proteolysis

To obtain more direct evidence regarding the specificity of the C5a serine protease, the effect of the purified protein was measured on a nonspecific substrate, ¹²⁵l-labelled bovine serum albumin (BSA). Nonspecific proteolysis was determined by comparing the inactivation of C5a with the hydrolysis of a similar quantity of ¹²⁵l-labelled BSA under identical experimental conditions. One-tenth μg of the protein of interest was maintained for 15 minutes at 37 °C with 5 μg purified C5a serine protease in 0.5 ml Dulbecco's phosphate-buffered saline (PBS). Loss of C5a was measured by the myeloperoxidase assay as described in Example 1B below (A^ in the absence of serine protease was 0.84). Proteolysis of BSA was stopped with 0.5 ml ice-cold unlabelled BSA (2 mg/ml) followed immediately by 0.2 ml 30% (w/v) trichloroacetic acid. After 10 minutes in ice, the precipitated proteins were removed by centrifugation for 10 minutes in an Eppendorf centrifuge. The radioactivity in 0.5 ml of each resultant supernatant was then measured in a gamma counter.

The results, shown in Table 2, confirm that the C5a serine protease is not a general protease that has wide or unlimited substrate specificity. Experiments with ¹²⁵l-labelled BSA contained an average of 1,458,700 cpm. The amount of ¹²⁵l released from the labelled albumin by trichloroacetic acid precipitation (the 0% value) was 135,700 +/- 4700 cpm before and 125,100 +/- 7500 cpm after treatment with the C5a serine protease.

Tablβ 2

Substrate Enzyme % degraded

E. Neutrophii Cell Chemotaxis Assays

Cell chemotaxis assays were used to determine the o presence of C5a serine protease in ascites fiuid and in ammonium sulfate precipitates. For the assay, ascites fiuid prepared as described in Example 1A was heated at 56 °C to inactivate complement, while the ammonium sulfate precipitates were dissolved in 10 mM Tris-HCl at pH 7.4 at a concentration corresponding to an absorbance at 280 nm of 6.6, 5 then desalted by dialysis against the same buffer. The C5a serine protease in the samples was determined by measuring the chemotaxis of freshly prepared human neutrophils in Boyden chambers by the leading front technique as described Zigmond et al., J. Exp. Med.. 137:387-410 (1973), using a mixture of 10% (v/v) sample and either 1% 0 (v/v) zymosan-activated serum or 1 nM recombinant C5a (rC5a) (Sigma Chemical Co.) as chemoattractant.

The neutrophils for use in the chemotaxis assays were isolated from blood collected after informed consent from medication-free normal volunteers. The blood was first anticoagulated with a mixture of 0.14 M citric acid, 0.2 M trisodium citrate, and 0.22 M dextrose. The anticoagulated blood was centrifuged at 800 x g for 15 minutes at room temperature and the platelet-rich plasma supernatant was discarded. The pelleted erythrocytes, mononuclear and polynuciear cells were resuspended and diluted with a volume equal to the starting blood volume with chilled 0.14 M PBS, pH 7.4. The peripheral blood mononuclear cells (PBMC) were depleted from the diluted cell suspension by centrifugation on low endotoxin Ficoll-Hypaque (Sigma Chemical. Co.) at 400 x g for 10 minutes at 18^βC.

The polynuciear cells (neutrophils) in the PBMC-depleted cell suspension were then recovered by dextran sedimentation. The resulting cell pellet containing neutrophils was resuspended at a concentration of 1.5 x 10⁷ cells/ml in calcium-free Hanks Balanced Salt Solution (HBSS) (Sigma Chemical Co.). The resuspended cell suspension was maintained on ice and used within two hours of isolation.

The material to be tested was place in the lower chamber of the Boyden apparatus below the Miilipore filter and the cells were placed in the upper chamber. After a suitable maintenance period, counts were made of the number of cells that have traversed the filter and were on the bottom side. An increase in the number was a measure of the chemotactic influence of the material in the lower chamber. Random migration was determined by measuring migration toward PBS containing 0.6% BSA and 0.1% glucose. Chemotaxis was calculated by subtracting the value for random migration from the distance in microns [micrometers (μm)] traveled in response to rC5a.

Table 3

Peritoneal fluid N Chemotaxis (μm) Inhibition of chemotaxis (%)

None 7 41.5 +/- 3 .8 0

Normal, untreated 8 24 . 1 +/- 6.5 53 .5 +/" ⁷ - ⁴ Normal, non-immune 3 24 .1 +/" 4 .2 53 .5 +/- 4. 6 serum-treated

Normal, antiserum- 3 46.1 +/- 2.0 -12.7 +/- ⁶ - ⁷ treated FMF 39.4 +/- 10. 6 5. 0 +/" ⁶ - ⁸

The effect of the rabbit antibody raised against C5a serine protease on the chemotaxis of neutrophils was also evaluated. For this aspect, the antiserum and the nonimmune normal serum were heat-inactivated at 56 "C for 30 minutes and diluted 1 :100 with PBS followed by maintenance with an equal volume of the peritoneal fluid for 30 minute at 37 ° C before the chemotaxis assay was performed. Random migration was 35.0 +/- 3.2 μm. Results are expressed as the mean +/- standard error of the mean. "N" indicates the number of experiments, each using a different heat inactivated peritoneal fluid.

Untreated or nonimmune serum-treated peritoneal fluid inhibited rC5a-mediated chemotaxis by greater than 50% confirming that an inhibitor of rC5a is present in peritoneal fluid. The effect was negated in the presence of antibodies against the C5a serine protease. In peritoneal fluid taken from familial Mediterranean fever (FMF), no inhibition of chemotaxis was observed thus confirming that this patient population lacks the C5a serine protease. Treatment of normal peritoneal fluid with the antiserum reduced the C5a serine protease activity to the levels found in peritoneal fluids from FMF patients. In addition, C5a serine protease had no effect on the chemotactic activity of the peptide substrate N- formyl-Methinoyl-Leucyl-Phenylalanine adding further support to the substrate specificity of the C5a serine protease.

F. Summary of the Characterization of Purified C5a Serine Protease

For use in the present invention, the purification of a C5a serine protease from ascites fluid is described. This C5a serine protease, previously demonstrated in near-normal synovial fluid and normal peritoneal fluid was purified to apparent homogeneity using an arginine agarose affinity column. This column was selected under the assumption that, like other serine proteases that participate in responses to tissue injury (e.g., certain clotting factors and fibrinolytic agents), the C5a serine protease might cleave C5a on the carboxyi side of an arginine residue. The specific activity of the pure enzyme was 3,000 times greater than that of the starting peritoneal fluid, suggesting that the enzyme is present in the peritoneal fluid at a concentration of approximately 1 mg/L. A high degree of substrate specificity for the C5a serine protease is thus suggested by a number of observations, including the findings that the protease converted C5a to a well-defined inactive fragment that was slightly smaller than C5a and was recognized by a polyclonal anti-C5a antibody, and that the partly purified enzyme, though able to cleave the chromogenic substrate S-2251 , could not hydrolyze S-2288. The C5a serine protease is not a general protease as it did not cleave BSA as shown in Table 2.

This antiserum raised in rabbits against the purified C5a serine protease recognized the 53-56 kDa band on a Western blot of partially purified C5a protease, while normal rabbit serum did not react. The antiserum did not recognize the protein on a Western blot of unfractionated peritoneal fluid, presumably because the amount was too low to be detected. It did, however, inhibit C5a inactivation by such fluids, while normal rabbit serum had little effect. Treatment of normal peritoneal fluids with the antiserum reduced C5a protease activity to the levels found in peritoneal fluids from patients with FMF as shown in Table 3.

The kinetic behavior of the C5a protease was evaluated by the spectrophotometric assays described in Example 1B, which allowed measurements to be made of initial rates of C5a inactivation. With this method, C5a inactivation was found to be proportional to the concentration of protease. The purified protease obeyed saturation kinetics as the concentration of C5a was varied, showing an apparent K^. for C5a of 0.96 μM and a V at 25° C of 33 nmoies C5a/min/μg protein. ln defining the physiological function of this serine protease, its substrate specificity is a key consideration. Earlier experiments showed that even after inactivation by this serine protease, C5a was recognized by a polyclonal anti-C5a antibody. This finding plus others discussed above strongly suggested that the range of substrates for this serine protease was very narrow. The results with ¹²⁵l-labelled BSA provide further support for this idea, and further suggest that the activity of the serine protease may be limited to few substrates the nature of which was not predictable.

Chemotactic inhibitors from other sources have been described by several investigators, but the C5a serine protease from peritoneal and synovial fluid of normal persons seems to be different from all of these. Its size and target of inhibition distinguishes it from cell-directed inhibitors such as polymeric IgA, the material responsible for impaired chemotaxis in cirrhotics [Van Epps et al., Am. J. Med.. 59:200-207 (1975)], and leukocyte inhibitory factor, a 70 kD antichemotactic protein released from stimulated lymphocytes [Rocklin, J. Immunol.. 114:1161-1165 (1975)]. It is distinguished from the chemotactic factor inactivators of serum [Ward et al., J. Clin. Invest. 58:123-129 (1969) and Till et al., J. Immunol.. 114:843-847 (1975)], tissue extracts [Bronza et al., J. Clin. Invest.. 56:616-623 (1975)] and the azurophil granules of neutrophils {Wright et al., J. Immunol.. 119:1068-1076 (1977) and Wright et al., Inflammation. 1:23-40 (1975)] by its stability at 56 ° C, a temperature at which those inactivators are rapidly destroyed. It is similar in size to the C5a inhibitor described in sera of patients with systemic lupus erythematosus, but the latter acts on the C5a cochemotaxin [Perez et al., J. Clin. Invest. 62:29-38 (1978) and Perez et al., J. Immunol.. 126:800-804 (1981)], while the C5a serine protease described herein cleaves C5a and C5a_dθ8arg directly.

The C5a serine protease described herein was found to be deficient in serosal fluids of patients with FMF, a disorder characterized by inappropriate inflammatory responses. The clinical features of FMF suggest that the function of the C5a serine protease is to prevent the development of unprovoked inflammatory reactions by counteracting the effects of small amounts of C5a that may be accidentally released from time to time into the serosal spaces. According to this formulation, the inappropriate inflammatory reactions typical of FMF would be due to inadequate suppression of accidentally released chemoattractant in the absence of this serine protease.

3. Inhibition of IL-8-Mediated Inflammation bv C5a Serine Protease

A. Inhibition of Neutrophii Chemotaxis: Dependence of IL-8 Inactivation on the Time of Exposure to Peritoneal Fluid

The protection afforded by the C5a serine protease against the development of an inflammatory reaction seen in normal subjects compared with that seen in patients with familial Mediterranean fever (FMF) suggested that this serine protease might have anti-inflammatory actions beyond its ability to destroy C5a. To verify this unexpected activity, the effect of the serine protease on the chemotactic activity of IL-8 was examined in neutrophii chemotaxis assays performed as described in Example 2E. In vivo. IL-8, which is produced by mononuclear phagocytes and synovial and serosal fibroblasts at sites of inflammation as well as fibroblasts and Type II epithelial cells in the lungs, acts on neutrophils in several ways, serving as a chemotaxin, stimulating 0₂- production and neutrophii degranulation, and increasing the expression of integrins and the complement receptor CRI on the neutrophii surface.

IL-8 is capable of maintaining an inflammatory reaction over time, a capability perhaps explained by the resistance of IL-8 to proteolysis and other environmental factors (e.g., pH and temperature), which allows it to remain active and attract neutrophils to a site of inflammation for many hours. In addition, the inflammatory response is a self-amplifying one in which the migration of neutrophils to an inflammatory site in response to chemotaxins results in the ultimate production of more chemotaxins and unregulated neutrophii accumulation. Thus, the discovery that IL-8-mediated inflammation was inhibited by C5a serine protease has therapeutic value for controlling inflammatory responses especially seen in distinct pulmonary diseases.

The initial experiments were performed with peritoneal fluid as prepared in Example 1A as the source of the C5a serine protease and recombinant human IL-8 as the target substrate. The monocyte-derived recombinant IL-8 having 72 amino acid residues was obtained from Genzyme, Cambridge MA.

The dependence of IL-8 inactivation on the time of exposure with peritoneal fluid was determined in neutrophii chemotaxis assays where peritoneal fluid (10% (v/v)) in PBS containing 1 mg/ml glucose and 6 mg/ml BSA (PBS+) was maintained with 50 ng/ml (6nM) IL-8 for different time intervals at 37 ° C. After each maintenance period, 3 mM PMSF was admixed to stop the reaction and the pH was then corrected to 7.4 before ceil admixture by using the same solutions. Chemotaxis was then measured in Boyden chambers as described in Example 2E and by Zigmond et al., J. EXP. Med.. 137:387-410 (1973). The vaiue for control IL-8 induced chemotaxis (corrected for random migration of 56.3 +/- 3.2 μm) was 46.2 +/- 2.9 μm. The results are expressed as the mean +/- one standard error of three experiments.

As shown in Figure 1 , the chemotactic activity of IL-8 was rapidly lost when the interleukin was maintained with peritoneal fluid prior to exposure to the neutrophils. Under the conditions of the experiment, IL-8 was extensively inactivated by 8 minutes, a rate comparable to that found for the inactivation of C5a by peritoneal fluid as shown in Example 1.

B. Inhibition of Neutrophii Chemotaxis: Dependence of IL-8

Inactivation as a Function of Peritoneal Fluid Concentration

To determine the effective concentration of the peritoneal fluid required to inactivate IL-8-mediated chemotaxis that leads to an inflammatory response in vivo, neutrophii chemotaxis assays were performed as described above in the presence of peritoneal fluid in concentrations ranging from 1 to 10%. Peritoneal fiuid diluted in PBS+ to the concentrations indicated was maintained for 10 minutes at 37 ° C with 50 ng/ml IL-8, after which chemotaxis was measured as described in Example 2E. The values for chemotaxis were corrected for random migration of 29.3 +/- 0.8 μm. The results are expressed as the mean +/- one standard error of three experiments.

The results, shown in Figure 2, reveal that IL-8 inactivation, as measured by less cell motility or chemotaxis, increased with increasing concentrations of peritoneal fluid. The most effective concentration of peritoneal fluid was 10% where the chemotaxis was less than 35 μm in distance. Thus, the inhibition of IL-8-mediated chemotaxis is both time and dose dependent as shown in Examples 3A and 3B.

C. Inhibition of Neutrophii Chemotaxis: Effect of IL-8

Concentration in the Presence and Absence of Peritoneal Fluid

To determine the dose response of increasing concentrations of IL-8 and inhibition of IL-8 mediated chemotaxis, assays were performed in the presence and absence of peritoneal fluid. Peritoneal fluid [3% (v/v)] in PBS+ was maintained with IL-8 ranging in concentration from 2 to greater than 100 ng/ml for 10 minutes at 37 °C after which chemotaxis was measured as described in Example 2E. For each assay performed with peritoneal fluid, a control assay was carried out under the same conditions except that the peritoneal fluid was replaced by an equal volume of buffer. The values for chemotaxis were corrected for random migration of 45.3 +/- 1.9 μm. The results are expressed as the mean +/- one standard error of three experiments.

Chemotaxis was inhibited when IL-8 concentrations as high as 0.1 μg/ml (100 ng/ml or 12 nM) were exposed to peritoneal fluid as shown in the line indicated by closed circles in Figure 3. No inhibition of chemotaxis was detected when IL-8 was exposed to buffer prior to being placed in the Boyden cnambers. These results are similar to those previously obtained with C5a as shown in Example 2. Thus, the inhibition of IL-8-mediated chemotaxis in vitro by C5a serine protease present in 3% peritoneal fiuid directly correlates to the inflammatory response seen ID vivo where the neutrophil-activating properties of IL-8 occur between 0.1 and 1.0 nM.

D. Inhibition of Neutrophii Chemotaxis: Effect of IL-8-Mediated

Chemotaxis in the Presence of Treated Peritoneal Fluid To confirm the identification of the IL-8 serine protease in peritoneal fluid preparations, several neutrophii chemotaxis assays were performed as described in Examples 2E and 3A in the presence of various treated peritoneal fluid preparations. Those included the following: 1 mM PMSF, a serine protease inhibitor; heated peritoneal fluid (either at 56 °C for 30 minutes or boiled) to determine if the inactivator was temperature sensitive; antibody-treated with an antibody raised against the C5a serine protease prepared in Example 2B; and lastly with

FMF-derived peritoneal fluid as described in Example 2E.

For the assays, PMSF-treated peritoneal fluid was prepared by treating peritoneal fluid diluted 1 :10 (v/v) in (PBS+) in a total volume of

0.3 ml with 3 mM PMSF for 15 minutes at 37 ° C. Heated peritoneal fluid was maintained at 56 ° C for 30 minutes and boiled fluid was prepared by heating in boiling water for 3 minutes, then centrifuging at 800 x g for 10 minutes to remove denatured proteins. Peritoneal fluid was treated with antiserum against the C5a serine protease or non-immune serum prepared in Example 2B that was diluted 1 :30 in PBS and used as previously described in Example 2E. The peritoneal fluids (10% (v/v) in

PBS) were maintained with 50 ng/ml (6nM) IL-8 for 10 minutes at 37° C, after which chemotaxis was measured in Boyden chambers as described in Example 2E. All chemotaxis values were corrected for random migration, which was 40.5+/- 2.5, 47.1 +/- 0.2 and 38.0 +/- 4.5 μm, respectiveiy, in Experiments 1 , 2 and 3. Results are expressed as the mean +/- one standard error for 3 determinations. Inhibition of chemotaxis was calculated as follows: percent inhibition = 100 X [1- (chemotaxis in the presence of the inactivator/ chemotaxis in the absence of the inactivator)].

These experiments indicate that the activity in peritoneal fluid responsible for the loss of the chemotactic potency of IL-8 was the same as that which destroys C5a as shown in Table 4. Like the C5a serine protease characterized in Example 2, the activity that destroyed IL-8 was a serine protease, as indicated by the sensitivity of IL-8 inactivation to PMSF, and was stable to heating at 56 °C for 30 minutes but not to boiling (Table 4, Experiment 1). IL-8 inactivation as measured by an decrease in chemotaxis was partly prevented by a polyclonal antibody that had been previously raised against the purified C5a serine protease (Table 4, Experiment 2). As with C5a, the chemotactic activity of IL-8 was not affected by peritoneal fluid from a patient with FMF (Table 4, Experiment 3) confirming that the serine protease that inhibits C5a missing in FMF patients is the responsible agent for inhibiting IL-8. Finally, the C5a and IL-8 serine proteases purified in parallel.

SUB TIT E SHEET Table 4

Chemotaxin Migration μ % of contro

Experiment 1

IL-8

IL-8 + PMSF

IL-8 + peritoneal fluid IL-8 + PMSF-treated -

peritoneal fluid

IL-8 + 56^βC peritoneal fluid 9.5 +/- 4.5 44 IL-8 + boiled peritoneal fluid 47.4 +/- 6.7 106 Experiment 2 IL-8 51.3 +/- 4.7 100 IL-8 + peritoneal fluid 11.7 +/" 3.3 27

IL-8 + peritoneal fluid + 14.7 +/- 2.4 28 non-immune seirum IL-8 + peritoneal fluid + 34.9 +/- I-⁷ 68 anti-C5a serine protease antiserum

IL-8 + anti-C5a serine protease 50.0 +/- 6.2 97 antiserum Experiment 3 IL-8 47.0 +/" 7.3 100 IL-8 + normal peritoneal fluid 19.0 +/- 8-6 40 IL-8 + FMF peritoneal fluid 50.7 +/- 7.6 108

SUBSTITUTESHEET Additional assays as described above were performed in the presence of the monoclonal antibodies prepared in Example 2B2) against purified C5a serine protease. As shown for the polyclonal antiserum, IL-8 inactivation as measured by an decrease in chemotaxis was prevented when the peritoneal fluid containing the C5a protease was pretreated with the anti-C5a serine protease monoclonal antibody. The C5a protease inactivated by the monoclonal antibody thus could not inactivate the IL-8 mediated chemotaxis. Since the polyclonal and monoclonal anti-C5a serine protease preparations inhibit the C5a protease which inactivates IL-8 in addition to C5a, they are thus useful for methods for ameliorating an IL-8 inactivating agent-mediated disorder where it would be beneficial to promote an inflammatory response. In addition, the anti-C5a serine protease preparations would allow for a method of detecting the presence of the IL-8 inactivating agent having the activity of a C5a serine protease in a body fluid sample. This would be especially beneficial to determine the presence of the inactivating agent in disease conditions such as FMF, adult respiratory distress syndrome, idiopathic pulmonary fibrosis and the like.

E. IL-8 Receptor Binding Assay An IL-8 receptor binding assay is performed to measure the effect of an IL-8 inactivating agent having the activity of C5a serine protease. For the assay, IL-8 receptor expressing neutrophils are prepared as described in Example 2E. Purified IL-8 obtained from Genzyme is first labeled with sodium ¹²⁵l (New England Nuclear, Boston, MA) by using the solid phase lactoperoxidase-giucose oxidase method as provided in an Enzymobead radioiodination reagent kit (Bio-Rad, Richmond, VA). To assay the inactivating activity of C5a serine protease on IL-8, either peritoneal fluid from 1 to 10% or 35 μl of 35 μg/ml concentration of purified C5a protease are admixed with 10 ng/ml ¹²⁵l-labelled IL-8 to form an admixture and maintained for 15 minutes at 22° C in a microfuge tube. The prepared 2 X 10⁵ IL-8 receptor-expressing neutrophils are then admixed with the labelled admixture in a final volume of 200 μl and maintained for 20 minutes at room temperature. The reaction admixture is then centrifuged at 11 ,000 X g for 30 seconds and subsequently 100 μl of the resultant supernatant is transferred to another tube and the radioactivity in both tubes, cell pellet plus supernatant versus supernatant alone, is determined in a gamma counter. The amount of ¹²⁵l-labelled-IL-8 bound to the receptors is then calculated as described by Ayesh et al., J. Immunol.. 144:3066-3070 (1990) and compared to control assays where the labeled ligand is maintained with buffer and not C5a serine protease. Since the activity of the C5a serine protease on IL-8 results in the inability of IL-8 to bind to its receptor, the presence of the C5a serine protease in a body fluid is determined by measuring a decrease in the binding of the labelled ligand to its receptor. In the case of an FMF patient, the absence of the C5a serine protease would result in binding of iodinated IL-8 equivalent to that of control.

F. Structural Analysis of IL-8 Treated with C5a Serine Protease

The effect of the serine protease on the structure of IL-8 was investigated in an experiment in which the interleukin was treated with the purified serine protease and the product examined by SDS-PAGE as described in Example 1B. IL-8 (1 μg/10 μl) (Genzyme) was premaintained with either 30 μl PBS or 30 μl of purified C5a serine protease prepared in Example 1B (35 μg/ml) for 20 hours at room temperature. The sampies were then subjected to SDS-PAGE under reducing conditions on 18% SDS-PAGE followed by staining with Coomassie Blue.

As shown in Figure 4 in the lanes labelled as A, control PBS-treated IL-8 migrated as 2 narrow bands that represented the interleukin monomer and dimer, as previously reported by Baggiolini et al., J. Clin. Invest.. 84:1045-1049 (1989), Schroder et al., J. Immunol.. 144:2223-2232 (1990) and Peveri et al., J. Exp. Med.. 167:1547-1559 (1988). The C5a serine protease-treated IL-8, however, showed only the monomer band, which was somewhat broader and migrated slightly more rapidly than control IL-8 as shown in lane B.

The foregoing data shows that a single anti-inflammatory serine protease of this invention is able to inactivate both C5a and IL-8, two chemotactic factors with little sequence homology. Precedent for this situation exists in the coagulation cascade, where thrombin, for example, proteolytically activates several procoagulant molecules of widely varying structure as described by Coughlin et al., J. Clin. Invest.. 89:351 (1992). IL-8 is recognized as an unusually stable inflammatory mediator and this is the first description of an serine protease capable of the rapid and efficient inactivation of the IL-8 chemotactic factor. Since C5a and IL-8 are important for both the initiation and the maintenance of inflammatory reactions, the serine protease present in peritoneal fluid, serosal fluid and the like, which inactivates both of these inflammatory mediators, is likely to serve a significant function as a regulator of inflammation in serosal spaces. Thus, the C5a serine protease is also an IL-8 inactivating agent as described in this invention.

In particular, in the lung where IL-8 is produced by mononuclear phagocytic cells as well as fibroblasts and Type II epithelial cells, IL-8 mediates different types of pulmonary inflammatory responses in diseases such as adult respiratory distress syndrome, idiopathic pulmonary fibrosis and late phase asthma, all of which are diseases which exhibit a significant neutrophii component. A preferred aspect of this invention is the C5a serine protease itself and its ability to alleviate of the IL-8 self-amplifying inflammatory cascade phenomenon characterized by the ultimate production of neutrophii chemotactic factors and uncontrolled migration of neutrophils into the inflammatory site.

The foregoing specification, including the specific embodiments and examples, is intended to be illustrative of the present invention and is not taken to be limiting. Numerous other variations and modifications can be effected without departing from the true spirit and scope of the present invention.

Claims

CLAIMS:

1. A method for inhibiting IL-8-mediated inflammation comprising administering a therapeutically effective amount of IL-8 inactivating agent or functional equivalents thereof in a pharmaceutically acceptable excipient wherein said agent has the activity of C5a serine protease.

2. The method of claim 1 wherein said composition is administered to said patient at a dose from 5 μg/hour to about 5 mg/hour.

3. The method of claim 1 wherein said composition is in the form of an aerosol for inhalation.

4. The method of claim 1 wherein said composition is in the form of an injectable solution for injection.

5. The method of claim 1 wherein said composition is taken orally.

6. The method of claim 1 wherein said composition is in the form of a suppository.

7. The method of claim 1 wherein said composition is in the form of an enema.

8. A method for detecting the presence of IL-8 inactivating agent which comprises contacting a sample suspected of containing said agent with a diagnosticaliy effective amount of IL-8 susceptible to inactivation by said agent.

9. The method of claim 8 wherein said agent has the activity of C5a serine protease.

10. The method of claim 8 wherein said detecting comprises measuring the activity of any IL-8 present after said contacting.

11. The method of claim 10 wherein said measuring is selected from the group consisting of: a cell chemotaxis assay; a myeloperoxidase release assay; a spectrophotometric assay; an IL-8 receptor binding assay; and gel electrophoresis.

12. The method of claim 8 wherein said sample is blood, serosal fiuid, peritoneal fluid, pleural fiuid, bronchoalveolar lavage fluid and synovial fluid.

13. The method of claim 8 wherein said sample is from a patient with familial Mediterranean fever.

14. A kit useful for the detection of IL-8 inactivating agent from a specimen, the kit comprising a package containing, in an amount sufficient for at least one assay, IL-8.

15. A therapeutic composition suitable for inhibiting IL-8-mediated inflammation comprising a therapeutically effective amount of IL-8 inactivating agent or functional equivalents thereof in a pharmaceutically acceptable excipient wherein said agent has the activity of C5a serine protease.