WO1993009803A1

WO1993009803A1 - Factor x-derived polypeptides and anti-peptide antibodies, systems and therapeutic methods for inhibiting inflammation

Info

Publication number: WO1993009803A1
Application number: PCT/US1992/010068
Authority: WO
Inventors: Dario C. Altieri; Thomas S. Edgington; Daryl S. Fair
Original assignee: The Scripps Research Institute; SCHAFFER, Susan, C.
Priority date: 1991-11-22
Filing date: 1992-11-20
Publication date: 1993-05-27
Also published as: AU3221793A

Abstract

The present invention describes Factor X-derived polypeptides and anti-polypeptide antibodies, and compositions thereof, capable of inhibiting Factor X binding to cells bearing the Mac-1 receptor, such as monocytes and macrophages, and thereby inhibit Mac-1 mediated procoagulant activity and associated inflammatory processes. Methods of inhibiting Mac-1-mediated inflammation in patients using the Factor X-derived polypeptides or antibodies. Also described are methods of inhibiting the interaction of leukocytes with vascular endothelium thereby regulating the consequent hemostatic, angiogenic, immunoproliferative and extravasational responses.

Description

FACTOR X-DERIVED POLYPEPTIDES AND

ANTI-PEPTIDE ANTIBODIES, SYSTEMS AND THERAPEUTIC

METHODS FOR INHIBITING INFLAMMATION

5 Description

Technical Field

The present invention relates to polypeptides and anti-peptide antibodies useful in therapeutic methods and compositions for inhibiting inflammation, related 10 Mac-l receptor-mediated procoagulant processes and

Mac-1 receptor-mediated interactions with endothelial cells.

Background 15 The macrophage/monocyte adhesive receptor Mac-1 participates in the process of cellular initiation of Factor X into the active coagulation protease Factor Xa in what is now known as a cellular pathway for initiation of the coagulation protease cascade. The 20 process includes a binding event between Factor X and the Mac-1 receptor (Altieri et al, J. Biol. Chem.. 263:7007-7015, 1988), followed by the activation of Factor X to Xa and assembly with other blood coagulation components to form a functional 25 prothrombinase complex (Altieri et al, Proc. Natl. Acad. Sci. USA. 85:7462-7466, 1988).

The initiation of the coagulation cascade by the cellular pathway invokes cellular immune inflammatory responses, and is an important pathway in inflammatory 30 processes distinct from the intrinsic or extrinsic pathways for the coagulation cascade.

Polypeptides derived from the sequence of Factor X active as inhibitors that block Factor X activity or !j.>, activation have been previously described. 35 Chattopadhyay et al, J. Biol. Chem. , 264:11035- (1989) . However the regions of Factor X involved in binding to Mac —1 receptor are not known, and the reported polypeptides have not been shown to inhibit the binding of Factor X to Mac-1. Factor X has also been shown to bind to the Herpes Simplex virus (HSV) glycoprotein gC when endothelial cells were infected by HSV. Etingin et al, Cell, 61:657-662 (1990). The HSV-infected cells were shown to bind Factor X and promote activation of Factor X to Xa, and subsequently provide a site for the assembly of a prothrombinase complex and the production of thro bin in the vicinity of the prothrombinase comp1e .

Brief Summary of the Invention

It has now been discovered that polypeptides derived from three different regions of Factor X have been identified that define the binding interaction between Factor X and the Mac-1 receptor (Mac-1) , and that these polypeptides can inhibit the binding of Factor X to Mac-1, thereby inhibiting the processes mediated by a Factor X: ac-1 complex.

Thus, the present invention contemplates a variety of Factor X-derived polypeptides from about 8 to 50 amino acid residues in length that have the capacity to inhibit Factor X binding to Mac-1 and include an amino acid residue sequence that defines a recognition site on Factor X for binding Mac-1 that is essential for activity of the Factor X: ac-1 complex. In a related embodiment, the present invention contemplates an antibody comprising antibody molecules that inhibits Factor X binding to Mac-1, and that immunoreact with a polypeptide described herein that is derived from Factor X that defines a recognition site on Factor X. Also described is a method of inhibiting Factor Xa-mediated monocyte procoagulant activity in a patient comprising administering to the patient a physiologically tolerable composition comprising a therapeutically effective amount of a Factor X-derived polypeptide or antibody that immunoreacts with a Factor X recognition site as described above.

Also contemplated is a method of inhibiting the interaction of leukocytes with endothelial cells thereby regulating leukocyte/endothelial cell-mediated responses in a patient comprising administering to said patient a physically tolerable composition comprising a therapeutically effective amount of a Factor X polypeptide that binds with a Factor X recognition site as described above. Those responses are not limited to inflammation but include leukocyte recruitment, leukocyte adhesion and extravasation, hematopoiesis, antigen presentation, angiogenesis, syncytial formation, and hemostasis.

Brief Description of the Drawings

In the drawings, forming a portion of this disclosure:

Figure 1 illustrates the effects of Factor X synthetic peptides on ¹⁵I-Factor X binding to monocyte THP-1 in Figure 1A to or HSV-infected endothelial cells in Figure IB.

The experimental conditions for ¹²⁵I-Factor X binding to monocyte THP-1 cells are described in Example 3a(3) . ¹²⁵I-Factor X binding to HSV-infected endothelial cells was performed as described in Example 4a. Increasing concentrations of the three Factor X peptides were separately admixed with 10 μM fMLP-stimulated THP-1 cells (A) or HSV-infected endothelial cells (B) and 15 nM ¹⁵I-Factor X. At equilibrium, specific binding was calculated as described in Example 3a(1) . For both A and B, polypeptide 366-373* (SEQ ID NO 18) is indicated by open circles, polypeptide 1:422-430 is indicated by open squares and polypeptide 230-246* (SEQ ID NO 7) is indicated by open triangles.

The data is plotted as the % of control ¹²⁵I- Factor X bound to either THP-1 cells (A) or HSV- infected endothelial cells (B) on the Y-axis against increasing concentrations of polypeptide x 10^"6 M plotted logarithmically on the X-axis.

Detailed Description of the Invention A. Definitions Amino Acid Residue: An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues identified herein are preferably in the natural "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem. _f 243:3552-59 (1969) and adopted at 37 CFR §1.822(b) (2) , abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE SYMBOL AMINO ACID

1-Letter 3-Letter

Y Tyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Ala alanine S Ser serine I He isoleucine L Leu leucine T Thr threonine V Val valine P Pro proline K Lys lysine H His histidine

Q Gin glutamine E Glu glutamic acid Trp tryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cys cysteine B Asx Asn or Asp Z Glx Gin or Glu X Xaa any amino acid

It should be noted that all amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. In addition, the phrase "amino acid residue" is broadly defined to include modified and unusual amino acids, such as those listed in 37 CFR §1.822 (b) (4) , and are incorporated by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates either a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group.

Antibody: The term antibody in its various grammatical forms is used herein to ref r to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab¹, F(ab')₂ and F(v) .

Antibody Combining Site: An antibody combining site is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) an antigen. The term immunoreact in its various forms means specific 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.

Anticoagulant: an agent that interrupts coagulation and thereby inhibits fibrin formation.

Coagulation: the sequential process in which the multiple coagulation factors of the blood interact resulting in the formation of fibrin.

Factor X: Factor X is a zy ogen of a serine protease which when activated and assembled into a prothrombinase complex functions in concert with Factor Va to cause the conversion of prothrombin to thro bin which promotes deposition of fibrin, chemotaxis of monocytes, platelets and leukocytes, and adhesion of macrophages to endothelium.

Monoclonal Antibody: The phrase monoclonal antibody in its various grammatical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific monoclonal antibody.

Polypeptide and Peptide: Polypeptide and peptide are terms used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.

Synthetic Peptide: Synthetic Peptide refers to a chemically produced chain of amino acid residues linked together by peptide bonds that is free of naturally occurring proteins and fragments thereof.

Zymogen: A precursor protein lacking enzymatic activity that is cleaved to yield an active protease. Conventional terminology identifies the active protein with a small "a": for example, Factor X is the zymogen and Factor Xa is the active protease.

B. Polypeptides

A polypeptide of the present invention has an amino acid residue sequence that includes a sequence derived from Factor X that corresponds to a region of Factor X that participates in the protein-protein binding interaction between Factor X and Mac-1. The region for protein-protein interaction is also referred to as a recognition site. Thus a polypeptide of this invention inhibits the binding of Factor X binding to Mac-1, and thereby inhibits the activity associated with a Factor X:Mac-1 complex present on the surface of cells that carry Mac-1, such as macrophages and monocytes. The activity associated with a Factor X: ac-1 complex on the surface of a macrophage or monocyte is referred to as monocyte procoagulant activity. This activity mediates the inflammatory processes of (1) localized thrombin production and subsequent deposition of fibrin in the vicinity of the complex, (2) chemotaxis of platelets, leukocytes and particularly macrophages to the complex, and (3) adhesion of macrophages to cells in the vicinity of the active complex. Because a polypeptide of this invention directly competes with Factor X for binding to Mac-1, that polypeptide inhibits binding of Factor X, and the associated monocyte procoagulant activity, thereby inhibiting inflammation mediated by the monocyte procoagulant activity associated with the Factor X:Mac-1 complex.

A polypeptide of the present invention is from 8 to 100 amino acid residues in length, preferably no more than about 50 residues and more preferably no more than about 20 residues in length, and includes an amino acid residue sequence defining a recognition site for Factor X binding to Mac-1 as described herein.

First Factor X Recognition Site

In one embodiment the invention contemplates a polypeptide that includes an amino acid residue sequence that defines a first recognition site of Factor X. The polypeptide includes an amino acid residue sequence represented by the formula -Gly-Tyr-Asp-Thr-Lys-Gln-Glu-Asp- that is shown in SEQ ID NO 1 from residues 366 to 373. Preferably, the polypeptide is no more that 12 amino acid residues in length. The SEQ ID NO and corresponding residues of a described amino acid residue sequence are conveniently recited herein in parenthesis after a designated amino acid residue sequence, where the first number is the SEQ ID NO and the range following the colon represents the residue numbers of the indicated amino acid residues in the sequence listing. For example, "(1:366-373)" refers to the sequence

"Gly-Tyr-Asp-Thr-Lys-Gln-Glu-Asp" shown in SEQ ID NO 1 from residue 366 to residue 373. A polypeptide defining the first recognition site of Factor X preferably has a sequence that corresponds to, and more preferably that is identical to, the sequence shown in SEQ ID NO 1.

Exemplary and preferred polypeptides defining a first Factor X recognition site have an amino acid residue sequence represented by a formula selected from the group consisting of Gly-Tyr-Asp-Thr-Lys-Gln- Glu-Asp-Gly (18:1-9), Gly-Tyr-Asp-Thr-Lys-Gln-Glu-Asp (1:366-373) and Phe-Cys-Ala-Gly-Tyr-Asp-Thr-Lys-Gln- Glu-Asp-Ala-Cys (1:363-375). Additional contemplated polypeptides have an amino acid residue sequence represented by a formula selected from the group consisting of Cys-Ala-Gly-Tyr-Asp-Thr-Lys-Gln-Glu-Asp- Ala-Cys (1:364-375), Ala-Gly-Tyr-Asp-Thr-Lys-Gln-Glu- Asp-Ala-Cys (1:365-375), Gly-Tyr-Asp-Thr-Lys-Gln-Glu- Asp-Ala-Cys (1:366-375) , Phe-Cys-Ala-Gly-Tyr-Asp-Thr- Lys-Gln-Glu-Asp-Ala (1:363-374), Cys-Ala-Gly-Tyr-Asp- Thr-Lys-Gln-Glu-Asp-Ala, (1:364-374) Ala-Gly-Tyr-Asp- Thr-Lys-Gln-Glu-Asp-Ala (1:365-374), Gly-Tyr-Asp-Thr- Lys-Gln-Glu-Asp-Ala (1:366-374), Phe-Cys-Ala-Gly-Tyr- Asp-Thr-Lys-Gln-Glu-Asp (1: 363-373 ) , Cys-Ala-Gly-Tyr- Asp-Thr-Lys-Gln-Glu-Asp (1: 364-373) and Ala-Gly-Tyr- Asp-Thr-Lys-Gln-Glu-Asp (1: 365-373) .

Second Factor X Recognition Site

In another embodiment the invention contemplates a polypeptide that includes an amino acid residue sequence that defines a second recognition site of Factor X. The polypeptide includes an amino acid residue sequence represented by the formula -Asp-Arg- Ser-Met-Lys-Thr-Arg-Gly- (1:423-430). Preferably, the polypeptide is no more than 14 amino acid residues in lengt .

A polypeptide defining the second recognition site of Factor X preferably has a sequence that corresponds to, and more preferably that is identical to, the sequence shown in SEQ ID NO 1.

Exemplary and preferred polypeptides defining a second Factor X recognition site have an amino acid residue sequence represented by a formula selected from the group consisting of Gly-Asp-Arg-Ser-Met-Lys- Thr-Arg-Gly (19:1-9), Ile-Asp-Arg-Ser-Met-Lys-Thr-Arg- Gly (1:422-430) , Asp-Arg-Ser-Met-Lys-Thr-Arg-Gly (1:423-430) and Ala-Phe-Leu-Lys-Trp-Ile-Asp-Arg-Ser- Met-Lys-Thr-Arg-Gly-Leu (1:417-431). Underlined amino acid residues indicate residues which do not correspond to the native Factor X sequence. Additional contemplated polypeptides have an amino acid residue sequence represented by a formula selected from the group consisting of Ile-Asp-Arg-Ser- Met-Lys-Thr-Arg-Gly-Leu (1:422-431) and Asp-Arg-Ser- Met-Lys-Thr-Arg-Gly-Leu (1:423-431).

Third Factor X Recognition Site Another embodiment of the invention contemplates a polypeptide that includes an amino acid residue sequence that defines a third recognition site of Factor X. The polypeptide includes an amino acid residue sequence represented by the formula -Leu-Tyr- Gln-Ala-Lys-Arg-Phe-Lys-Val- (1:238-246). Preferably, the polypeptide is no more than 25 amino acid residues in length.

A polypeptide defining the third recognition site of Factor X preferably has a sequence that corresponds to, and more preferably that is identical to, the sequence shown in SEQ ID NO 1.

Exemplary and preferred polypeptides defining a third Factor X recognition site have an amino acid residue sequence represented by a formula selected from the group consisting of Gly-Leu-Tyr-Gln-Ala-Lys- Arg-Phe-Lys-Val (7:1-10), Leu-Tyr-Gln-Ala-Lys-Arg-Phe- Lvs-Val-Glv (7:2-11), Gl -Leu-Tyr-Gln-Ala-Lys-Arg-Phe- Lvs-Val-Glv (7:1-11), Leu-Tyr-Gln-Ala-Lys-Arg-Phe-Lys- Val-Arg-Asn-Gly-Asp-Arg-Asn-Thr-Thr-Glu (8:1-18), Leu- Tyr-Gln-Ala-Lys-Arg-Phe-Lys-Val (1:238-246), Leu-Tyr- Gln-Ala-Lys-Arg-Phe-Lys-Val-Arg-Val-Gly-Asp-Arg-Asn- Thr-Glu-Gln (1:238-255) and Cys-Leu-Tyr-Gln-Ala-Lys- Arg-Phe-Lys-Val-Arg-Val-Gly-Asp-Arg-Asn-Thr-Glu-Gln- Glu-Glu-Gly-Gly-Glu-Ala-Val (1:237-262) . Additional contemplated polypeptides have an amino acid residue sequence represented by a formula shown by a SEQ ID NO selected from the group consisting of (1:237-262), (1:237-261), (1:237-260), (1:237-259), (1:237-258), (1:237-257), (1:237-256), (1:237-255), (1:237-254), (1:237-253), (1:237-252) , (1:237-251), (1:237-250) , (1:237-249), (1:237-248), (1:237-247), (1:237-246), (1:238-262), (1:238-261), (1:238-260), (1:238-259), (1:238-258) , (1:238-257) , (1:238-256), (1:238-255) , (1:238-254), (1:238-253) , (1:238-252), (1:238-251), (1:238-250) , (1:238-249), (1:238-248) , (1:238-247) and (1:238—246) .. Further contemplated are the above- recited polypeptides having one or more amino acid residue substitutions selected from the group consisting of Asn at position 248, Thr at position 254, and Glu at position 255.

The polypeptides described herein inhibit the binding of Factor X to Mac-1 by competing for the binding interaction at the recognition site from which the polypeptide was derived. Although binding at each recognition site is shown by the teachings herein to be independently inhabitable, combinations of inhibition directed to more than one recognition site can be accomplished by the use of combinations of polypeptides derived from more than one recognition site.

Thus, the present invention contemplates a composition as described further herein containing at least two different polypeptides corresponding to different recognitions sites of Factor X, each different polypeptide having an amino acid residue sequence as defined for a recognition site.

A polypeptide or combination of polypeptides of the present invention has the capacity to inhibit the binding of Factor X to Mac-1 as shown by the teachings herein, and thereby inhibit monocyte procoagulant activity and the associated inflammation processes.

Methods to measure the inhibition of a Factor X binding to Mac-1 are conveniently carried out in vitro in a standardized assay that measures the competitive inhibition of binding of Labelled Factor X to culture cells containing Mac-1. Exemplary assays for competitive inhibition of direct binding are described herein. In addition, functional assays indicative of inhibition of monocyte procoagulant activity are described that are suitable for measuring the inhibition by the polypeptides of this invention. A polypeptide is considered inhibitory where there is a decrease in the direct binding of labelled Factor X to cell surface Mac-1 in the presence of competing polypeptides in an amount greater than about 50 to 60 percent, preferably at least 60 percent, of control binding in the absence of peptide, and where the inhibition is titratable. Inhibition is titratable where there is a linear decrease in inhibition across a log or half log dilution series of polypeptide in the competition binding reaction.

Preferably, a polypeptide of this invention is further characterized by its ability to immunologically mimic an epitope (antigenic determinant) expressed by the recognition site from which the polypeptide was derived.

As used herein, the phrase "immunologically mimic" in its various grammatical forms refers to the ability of a polypeptide of this invention to immunoreact with an antibody of the present invention that immunoreacts with a native epitope on a recognition site as defined herein.

It should be understood that a subject polypeptide need not be identical to the amino acid residue sequence of Factor X, so long as it includes the required sequence and is able to inhibit binding as described herein.

A subject polypeptide includes any analog, fragment or chemical derivative of a polypeptide whose amino acid residue sequence is shown herein so long as the polypeptide is capable of inhibiting Factor X binding to Mac-1. Therefore, a present polypeptide can be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use. In this regard, a polypeptide of this invention corresponds to, rather than is identical to, the sequence of Factor X where one or more changes are made and it retains the ability to inhibit binding in one or more of the assays as defined herein.

The term "analog" includes any polypeptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the ability to inhibit binding 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 inhibition activity.

"Chemical derivative" refers to a subject 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 sulfonyl 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 O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 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 for serine; and ornithine may be substituted for lysine.

Particularly preferred modifications are those modifications designed to increase the stability of the polypeptide in solution, and therefore serve to prolong half life of the polypeptides in solutions, particularly biological fluids where proteases may be present such as in vivo in the bloodstream. Exemplary modifications are those that block susceptibility to proteolytic activity in the blood. Thus a polypeptide can have a stabilizing group at one or both termini. Typical stabilizing groups include amido, acetyl, benzyl, phenyl, tosyl, alkoxycarbonyl, alkyl carbonyl, benzyloxycarbonyl and the like end group modifications. Additional modifications include using a "L" amino acid in place of a "D" amino acid at the termini, cyclization of the polypeptide, and amide rather than amino or carboxy termini to inhibit exopeptidase activity.

Polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions or residues relative to the sequence of a polypeptide whose sequence is shown herein, so long as the requisite activity is maintained. The term "fragment" refers to any subject polypeptide having an amino acid residue sequence shorter than that of a polypeptide whose amino acid residue sequence is shown herein. When a polypeptide of the present invention has a sequence that is not identical to the sequence of Factor X, it is typically because one or more conservative or non-conservative substitutions have been made, usually no more than about 30 number percent, and preferably no more than 10 number percent of the amino acid residues are substituted.

"Substantially homologous" means that a particular subject sequence or molecule, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between reference and subject sequences. For purposes of the present invention, amino acid sequences having greater than 90 percent similarity, equivalent biological activity, and equivalent expression characteristics are considered substantially homologous and are included within the scope of a polypeptide of this invention.

Amino acid sequences having greater than 40 percent similarity are considered substantially similar. For purposes of determining homology or similarity, truncation or internal deletions of the reference sequence should be disregarded, as should subsequent modifications of the molecule, e.g., glycosylation. Sequences having lesser degrees of homology and comparable bioactivity are considered equivalents.

Additional residues may also be added at either terminus of an polypeptide of this invention 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 do not form epitopes which are cross reactive with Factor X, i.e., are not sufficiently similar in structure to a Factor X polypeptide as to produce cross-reacting antibodies.

Labels, solid matrices and carriers that can be used with the polypeptides 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, but do not form epitopes cross- reactive with a Factor X polypeptide. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. In addition, a subject polypeptide can differ, unless otherwise specified, from the natural sequence of the corresponding protease by the sequence being modified by terminal-NH₂ acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal- carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications.

When coupled to a carrier to form what is known in the art as a carrier-hapten conjugate, a polypeptide of the present invention is capable of inducing antibodies that immunoreact with the corresponding protease. In view of the well established principle of immunologic cross-reactivity, the present invention therefore contemplates antigenically related variants of a polypeptide of this invention. An "antigenically related variant" is a subject polypeptide that is capable of inducing antibody molecules that immunoreact with a polypeptide of this invention and immunoreact with Factor X. Any peptide 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, sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, aleic 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) . A polypeptide of the present invention also referred to herein as a subject polypeptide, can be synthesized by any of the techniques that are known to those skilled in the polypeptide art, including reco binant DNA techniques. Synthetic chemistry techniques, such as a solid-phase Merrifield-type synthesis, are preferred for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production and the like. An excellent summary of the many techniques available can be found in J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969; M. Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976 and J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York) , 1983 for solid phase peptide synthesis, and E. Schroder and K. Kubke, "The Peptides", Vol. 1, Academic Press (New York) , 1965 for classical solution synthesis, each of which is incorporated herein by reference. Appropriate protective groups usable in such synthesis are described in the above texts and in J.F.W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973, which is incorporated herein by reference. In general, the solid-phase synthesis methods contemplated comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine. Using a solid phase synthesis as exemplary, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to afford the final polypeptide.

A polypeptide of this invention can be used, inter alia, in the therapeutic methods of the present invention to inhibit the binding of Factor X to Mac-1 and thereby the attendant inflammation processes. A polypeptide can also be used to prepare an inoculum as described herein for the preparation of antibodies that immunoreact with epitopes in a recognition site of Factor X. In addition, a polypeptide can be used in vitro to inhibit binding of Factor X where binding is not desired.

C. Antibodies and Monoclonal Antibodies The term "antibody" in its various grammatical forms is used herein as a collective noun that refers to a population of immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.

An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen. The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules for use in the diagnostic methods and systems of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin 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 to Theofilopolous and Dixon. 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 are preferred, and are utilized as illustrative herein.

An antibody of the present invention comprises antibody molecules that immunoreact with a Factor X recognition site as defined herein and inhibit Factor X binding to Mac-1, thereby inhibiting the monocyte procoagulant activities described herein attributable to the formation of a cell surface Factor X:Mac-1 complex .

Antibody molecules of this invention are further characterized as being capable of immunoreacting with 1) isolated Factor X, and 2) a polypeptide of the present invention derived from one of the Factor X recognition sites, and being substantially free of antibody molecules that immunoreact with a polypeptide derived from a region of Factor X that is not within a recognition site as defined herein.

Thus in one embodiment, the invention contemplates an antibody comprising antibody molecules that inhibit Factor X binding to Mac-1 and that immunoreact with (a) Factor X, and with (b) a polypeptide having an amino acid residue sequence that includes a sequence represented by the formula shown in SEQ ID NO (1:366-373); but do not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula shown in the SEQ ID NO (1:174-201). This variety of antibody immunoreacts with the first recognition site of Factor X. Preferably, an antibody of this type also does not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula shown in the SEQ ID NO (1:374-390).

In another embodiment, the invention contemplates an antibody comprising antibody molecules that inhibit Factor X binding to Mac-1 and that immunoreact with (a) Factor X, and with (b) a polypeptide having an amino acid residue sequence that includes a sequence represented by the formula shown in SEQ ID NO (1:423- 430) ; but do not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula shown in SEQ ID NO (1:174-201). This variety of antibody immunoreacts with the second recognition site of Factor X. Preferably, an antibody of this "type also does not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula shown in the SEQ ID NO (1:404-422).

A related embodiment of the invention contemplates an antibody comprising antibody molecules that inhibit Factor X binding to Mac-1 and that immunoreact with (a) Factor X, and with (b) a polypeptide having an amino acid residue sequence that includes a sequence represented by the formula shown in SEQ ID NO (1:238-246); but do not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula shown in the SEQ ID NO (1:174-201). This variety of antibody immunoreacts with the third recognition site of Factor X. Preferably, an antibody of this type also does not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula shown in the SEQ ID NO (1:247-262).

Antibody immunoreactivity with the above- specified polypeptides and Factor X antigens can be measured by a variety of immunological assays known in the art. Exemplary immunoreaction of an antibody with isolated Factor X or polypeptides can be assayed at least by the methods described in the Examples. In addition, an antibody can be evaluated for its ability to inhibit Factor X binding to Mac-1 at least by the competition assay described in the Examples.

Exemplary anti-polypeptide antibodies having the above immunoreactivities are described in the Examples. By "substantially free" means that the antibody molecules do not immunoreact with the stated antigen at levels within one order of magnitude, and preferably within two orders of magnitude, of the levels of positive immunoreacting species of antigen. An antibody of the present invention is typically produced by immunizing a mammal with an inoculum containing a polypeptide of this invention and thereby induce in the mammal antibody molecules having immunospecificity for the recited polypeptide. The antibody molecules are then collected from the mammal and isolated to the extent desired by well known techniques such as, for example, by using DEAE Sephadex to obtain the IgG fraction. Exemplary antibody preparation methods are described herein. The preparation of antibodies against polypeptide is well known in the art. [See Staudt et al., J. Exp. Med.. 157:687-704 (1983)]. Briefly, to produce a peptide antibody composition of this invention, a laboratory mammal is inoculated with an immunologically effective amount of a recited polypeptide, typically as present in a vaccine of the present invention. The anti-polypeptide antibody molecules thereby induced are then collected from the mammal and those immunospecific for both Factor X and the immunizing polypeptide are isolated to the extent desired by well known techniques such as, for example, by immunoaffinity chro atography.

To enhance the specificity of the antibody, for example to specifically exclude immunoreactivity with particular polypeptides, the antibodies may be purified by immunoaffinity chromatography using solid phase-affixed immunizing polypeptide. The antibody is contacted with the solid phase-affixed immunizing polypeptide for a period of time sufficient for the polypeptide to immunoreact with the antibody molecules to form a solid phase-affixed immunocomplex. The bound antibodies are separated from the complex by standard techniques.

The word "inoculum" in its various grammatical forms is used herein to describe a composition containing a polypeptide of this invention as an active ingredient used for the preparation of antibodies against a polypeptide and Factor X. When a polypeptide is used in an inoculum to induce antibodies it is to be understood that the polypeptide can be used in various embodiments, e.g., alone or linked to a carrier as a conjugate, or as a polypeptide polymer. However, for ease of expression and in context of a polypeptide inoculum, the various embodiments of the polypeptides of this invention are collectively referred to herein by the term "polypeptide", and its various grammatical forms.

For a polypeptide 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.

One or more additional amino acid residues can be added to the amino- or carboxy-termini of the polypeptide to assist in binding the polypeptide to a carrier. Cysteine residues added at the amino- or carboxy-termini of the polypeptide have been found to be particularly useful for forming conjugates via disulfide bonds. However, other methods well known in the art for preparing conjugates can also be used. Exemplary additional linking procedures include the use of Michael addition reaction products, di- aldehydes such as glutaraldehyde, Klipstein, et al., J. Infect. Pis., 147:318-326 (1983) and the like, or the use of carbodiimide technology as in the use of a water-soluble carbodiimide to form amide links to the carrier. For a review of protein conjugation or coupling through activated functional groups, see Aurameas, et al., Scand. J. Immunol. , 1:7-23 (1978). Alternatively, the heterobifunctional cross-linker SPDP (N-succinimidyl-3-(2-pyridyldithio) proprionate)) can be used to conjugate peptides, in which a carboxy- terminal cysteine has been introduced.

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 polyamino acids such as poly (D-lysine: D-glutamic acid) , and the like.

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

The present inoculum contains an effective, immunogenic amount of a polypeptide of this invention, typically as a conjugate linked to a carrier. The effective amount of polypeptide per unit dose sufficient to induce an immune response to the immunizing polypeptide depends, among other things, on the species of animal inoculated, the body weight of the animal and the chosen inoculation regimen as is well known in the art. Inocula typically contain polypeptide concentrations of about 10 micrograms to about 500 milligrams per inoculation (dose) , preferably about 50 micrograms to about 50 milligrams per dose. The term "unit dose" as it pertains to the inocula refers to physically discrete units suitable as unitary dosages for animals, each unit containing a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent; i.e., carrier, or vehicle. The specifications for the novel unit dose of an inoculum of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active material and the particular immunologic effect to be achieved, and (b) the limitations inherent in the art of compounding such active material for immunologic use in animals, as disclosed in detail herein, these being features of the present invention. Inocula are typically prepared from the dried solid polypeptide-conjugate by dispersing the polypeptide-conjugate in a physiologically tolerable (acceptable) diluent such as water, saline or phosphate-buffered saline to form an aqueous composition. Inocula can also include an adjuvant as part of the diluent. Adjuvants such as complete Freund's adjuvant (CFA) , incomplete Freund's adjuvant (IFA) and alum are materials well known in the art, and are available commercially from several sources.

The techniques of polypeptide conjugation or coupling through activated functional groups presently known in the art are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol.. Vol. 8, Suppl. 7:7-23 (1978) and U.S. Patent No. 4,493,795, No. 3,791,932 and No. 3,839,153. In addition, a site directed coupling reaction can be carried out so that any loss of activity due to polypeptide orientation after coupling can be minimized. See, for example, Rodwell et al.,

Biotech.. 3:889-894 (1985), and U.S. Patent No. 4,671,958.

One or more additional amino acid residues may be added to the amino- or carboxy-termini of the polypeptide to assist in binding the polypeptide to form a conjugate. Cysteine residues, usually added at the carboxy-terminus of the polypeptide, have been found to be particularly useful for forming conjugates via disulfide bonds, but other methods well-known in the art for preparing conjugates may be used.

An antibody as described herein can be used, inter alia, in the diagnostic methods and systems of the present invention to detect the presence in a body sample of Factor X. A particularly preferred diagnostic method is to monitor the fate of a therapeutically administered polypeptide of this invention, using antibodies immunospecific for the polypeptide, as described herein.

An antibody of this invention can also be used in the therapeutic methods of the present invention to inhibit Factor X binding to Mac-1, and thereby inhibit Mac-1 mediated inflammation for the same conditions as discussed when an inhibitory polypeptide would be useful. A preferred antibody is a monoclonal antibody and is used herein as exemplary of an anti-polypeptide antibody of this invention.

The phrase "monoclonal antibody" in its various grammatical forms refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody.

A monoclonal antibody of this invention is further characterized as being capable of immunoreacting with 1) isolated Factor X, and 2) a polypeptide of the present invention as described for the antibodies of this invention.

A monoclonal antibody is typically composed of antibodies produced by clones of a single cell called a hybridoma that secretes (produces) only one kind of antibody molecule. The hybridoma cell is formed by fusing an antibody-producing cell and a myeloma or other self-perpetuating cell line. The preparation of such antibodies was first described by Kohler and

Milstein, Nature 256:495-497 (1975), which description is incorporated by reference. The hybridoma supernates so prepared can be screened for the presence of antibody molecules that immunoreact with the immunizing polypeptides or with the corresponding serine protease, or for inhibition of serine protease as described further herein.

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 an antigen, such as is present in a polypeptide of this invention. The polypeptide- induced hybridoma technology is described by Niman et al., Proc. Natl. Acad. Sci. USA. 80:4949-4953 (1983), which description is incorporated herein by reference.

It is preferred that the myeloma cell line used to prepare a hybridoma be from the same species as the lymphocytes. Typically, a mouse of the strain 129 G1X⁺ is the preferred mammal. Suitable mouse myelomas for use in the present invention include the hypoxanthine-aminopterin-thy idine-sensitive (HAT) cell lines P3X63-Ag8.653, and Sp2/0-Agl4 that are available from the American Type Culture Collection, Rockville, MD, under the designations CRL 1580 and CRL 1581, respectively.

Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 1500. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing a monoclonal antibody of this invention are identified using the radioimmunoassay (RIA) and the enzyme linked immunosorbent assay (ELISA) described in the Examples.

A monoclonal antibody of the present invention can also 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 antibodies of this invention can be used in the same manner as disclosed herein for antibodies of the present invention.

For example, the monoclonal antibody can be used in the therapeutic, diagnostic or in vitro methods disclosed herein where inhibition of Factor X binding to Mac-1 is desired.

Other methods of producing a monoclonal antibody, a hybridoma cell, or a hybridoma cell culture are also well known. See, for example, the method of isolating monoclonal antibodies from an immunological repertoire as described by Sastry, et al. , Proc. Natl. Acad. Sci.USA, 86:5728-5732 (1989); and Huse et al. , Science, 246:1275-1281 (1989).

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

D. Therapeutic Compositions

The present invention contemplates therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with an inhibitor of Factor X binding to Mac-1, namely a polypeptide, an anti-polypeptide antibody or monoclonal antibody as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic 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. A therapeutic composition can also be formulated for therapeutic administration as a tablet, pill, capsule, aerosol, sustained release formulation or powder. 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. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, 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.

Liquid 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 contains an amount of polypeptide or antibody of the present invention sufficient for convenient administration of a therapeutically effective amount, typically an amount of at least 0.1 weight percent of inhibitor per weight of total therapeutic composition. A weight percent is a ratio by weight of inhibitor to total composition. Thus, for example, 0.1 weight percent is 0.1 grams of inhibitor per 100 grams of total composition.

In one embodiment, a therapeutic composition can contain one or more species of polypeptide derived as described herein from a single Factor X recognition site, or can contain a combination of polypeptides derived as described herein from different recognition sites, which polypeptides are defined herein.

Thus, the invention contemplates a composition capable of inhibiting binding of Factor X to Mac-1 comprising at least two different polypeptides, each different polypeptide being as described herein and each including a sequence represented by a SEQ ID NO formula selected from different formulas in the group of formulas consisting of:

(a) (1:366-373), (b) (1:423-430), and

(c) (1:238-246). Polypeptides in group (a) are the polypeptides described herein derived from the first recognition site, group (b) are derived from the second recognition site and group (c) are derived from the third recognition site.

The relative amounts of the included polypeptides in a composition can vary widely because, as shown herein, polypeptides from each recognition site are independently effective at inhibiting Factor X binding to Mac-1. However, it is preferred that molar ratios of included polypeptides be in the range of 0.01:1, preferably 0.1:1, and more preferably in approximately equimolar (1:1) amounts. In another embodiment, a composition can contain one or more species of antibodies as defined herein.

E. Therapeutic Methods

1. Methods for Inhibiting Factor X binding to Mac-1. and Mac-1 Mediated Inflammation

It has been discovered that the polypeptides, antibodies, and monoclonal antibodies of the present invention have the capacity to inhibit Factor X binding to Mac-1, and thereby inhibit the inflammatory processes mediated by a Factor X:Mac-l complex, or monocyte procoagulant activity mediated processes.

Thus, the present invention provides for a method for inhibiting Mac-1 mediated inflammatory processes (monocyte procoagulant activity) in a patient comprising administering to the patient a therapeutically effective amount of an inhibitor of this invention, namely a polypeptide, antibody, or monoclonal antibody of the present invention in a physiologically tolerable therapeutic composition as described herein.

A therapeutically effective amount of an inhibitor is a predetermined amount calculated to achieve the desired effect, i.e., to inhibit in vivo the Factor X binding to Mac-1, and thereby reduce the Mac-1 mediated inflammation associated with monocyte procoagulant activity due to Factor X binding. The method can be practiced on a patient during an episode of inflammation to reduce ongoing inflammatory responses, or can be practiced on a patient as a prophylactic to inhibit future or anticipated inflammatory responses by administration of the therapeutic composition prior to the occurrence of the inflammatory response.

A typical clinical setting for in vivo inhibition of monocyte procoagulant activity is in a patient at risk for thromobis or an atherosclerotic event, such as a candidate for surgery, particularly major surgery, patients exhibiting disseminated intravascular coagulation (DIC) , septic shock, venous or arterial thrombosis, inflammation associated with bacterial or viral infections, inflammations of the cellular immune type such as arthritis, sarcoidosis, allergic encephalitis, and the like conditions, and patients undergoing delayed-type hypersensitivity reactions.

Inflammation in the occurrence of the above described clinical conditions is diagnosed by generally-accepted practice, and can include the secondary symptoms of fever and increased white blood cell counts (leukocytosis) , and can include the primary symptoms of localized edema, delayed type hypersensitivity and the like symptoms.

A therapeutically effective amount of a polypeptide of this invention is typically an amount of polypeptide such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma (intravascular) concentration of from about 0.05 micromolar (uM) to about 1000 Urn, preferably from about 0.1 uM to about 500 uM, and more preferably from about 1 uM to about 100 uM.

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 is sufficient to achieve a plasma concentration of from about 0.1 microgram (ug) per milliliter (ml) to about 100 ug/ml, preferably from about 1 ug/ml to about 5 ug/ml, and usually about 5 ug/ml.

A therapeutically effective level of inhibition of monocyte procoagulant activity in a patient indicative of the efficacy of inhibition therapy can be readily determined by a variety of routine clinical analyses to measure signs of monocyte procoagulant activity, and can further be assessed by monitoring the above described symptoms.

The therapeutic compositions containing a inhibitor of this invention are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, 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.

The compositions 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, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. A single injection is also referred to as a bolus injection. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

As an aid to the administration of effective amounts of a polypeptide, antibody, or monoclonal antibody of this invention, a diagnostic method for detecting a therapeutically administered polypeptide, antibody, or monoclonal antibody, respectively, in the subject's blood is useful to characterize the fate of the administered therapeutic composition. Suitable diagnostic methods are described herein.

In another embodiment, a therapeutic method of administering an inhibitor of this invention is by absorption into the skin or other tissue where inflammation occurs. Thus a cream, oil, ointment, suspension or other fluid or spreadable composition may be used that contains an effective amount of an inhibitor in a suitable carrier.

Alternatively, the therapeutic composition can be provided in a delivery device, such as a patch to be applied to the skin, or in a semipermeable container, designed for slow, measured release of the active compound into the adjacent tissues in contact with the delivery device, as are generally well known in the arts. Thus in one embodiment the invention contemplates a method for inhibiting Factor Xa-mediated monocyte procoagulant activity in a patient comprising administering to the patient a physiologically tolerable composition comprising a therapeutically effective amount of a Factor X polypeptide of this invention. Preferably the polypeptide has an amino acid residue sequence that includes a sequence represented by a formula shown in a SEQ ID NO selected from the group consisting of (1:366-373), (1:423-430) and (1:238-246) .

In one variation of this embodiment, the polypeptide has an amino acid residue sequence represented by a SEQ ID NO formula selected from the group consisting of (1:363-375), (18:1-9), (1:366- 373), (1:417-431), (19:1-9), (1:422-430), (1:423-430), (1:237-262), (7:1-10), (7:2-11), (7:1-11), (8:1-18), (1:238-246) and (1:238-255).

In another embodiment, the invention contemplates a method for inhibiting Factor Xa-mediated monocyte procoagulant activity in a patient comprising administering to the patient a physiologically tolerable composition comprising a therapeutically effective amount of an anti-Factor X polypeptide of this invention. The anti-Factor X polypeptide antibody comprises antibody molecules that inhibit Factor X binding to Mac-1 and that immunoreact with (a) Factor X, and with (b) a polypeptide having an amino acid residue sequence that includes a sequence represented by the formula shown in a SEQ ID NO selected from the group consisting of (1:366-373), (1:423-430) and (1:238-246); but do not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula shown in the SEQ ID NO formula (1:174-201).

2. Methods for Inhibiting Herpes Simplex Virus

Mediated Inflammation It has also been discovered, as shown by the teachings herein, that Factor X binds to Herpes Simplex Virus (HSV) glycoprotein C (gC) , which is expressed on the surface of at least endothelial cells during an HSV infection as a cell surface receptor. Thus, the polypeptides and antibodies of the present invention have the capacity to inhibit Factor X binding to gC cells in a manner analogous to their capacity to inhibit Factor X binding to Mac-1-bearing cells.

The clinical significance of the binding interaction between gC and Factor X is that HSV- infected endothelium, upon binding by Factor X, assembles a functioning prothro binase complex in a manner similar to a macrophage expressing Mac-1 complexed with Factor X. Etingin et al, Cell, 61:657 (1990) . HSV-infected endothelium, such as vascular tissue cell wall, exhibit procoagulant inflammatory activity that is similar to the processes associated with monocyte procoagulant activity. Thus, inhibiting the binding of Factor X to gC inhibits the inflammatory processes occurring locally in the vicinity of a HSV-infected endothelium.

HSV-infection mediated inflammatory processes occurring as a result of Factor X binding to gC expressed on endothelium include local generation of thrombin, local deposition of fibrin, increased monocyte adhesion to the infected endothelium and the pleiotropic effects associated with the monocyte adhesion. These pleiotropic effects include monocyte mediated damage to the endothelium and atherosclerotic plaque formation at the site of fibrin deposition. Thus the present invention contemplates, in one embodiment, a method for inhibiting HSV-mediated inflammation in the endothelial tissue of a HSV infected patient comprising contacting the cells of the endothelial tissue with a physiologically tolerable composition containing a therapeutically effective amount of a Factor X polypeptide, or anti- polypeptide antibody, of this invention, thereby inhibiting the binding of Factor X to any gC protein expressed on the surface of the HSV-infected endothelial cells.

Insofar as a particularly important endothelial tissue for the pathogenesis of HSV infection is the vascular system, and the gC protein is expressed on the surface of HSV-infected endothelium of the vascular system that faces inward contacting the blood stream, contacting the infected endothelial cells is most conveniently accomplished by intravenous administration of a therapeutic reagent of this invention. Such administration is as described before for inhibiting Factor X binding to Mac-1.

Insofar as Factor X binding to gC protein of HSV infected tissue mediates atherosclerotic plaque formation, the present invention also contemplates a method for inhibiting atherosclerotic plaque formation in an HSV-infected patient comprising administering to the vasculature of said patient by intravenous injection a physiologically tolerable composition containing a therapeutically effective amount of a Factor X polypeptide, or anti-polypeptide antibody, of this invention.

A therapeutically effective amount in this embodiment is generally as previously described for inhibiting Factor X binding to Mac-1, as are the modes of administering the therapeutic compositions, and the forms of the compositions. Additional teachings of dosages for effective inhibition can be found in the data described in the Examples.

3. Methods for Inhibiting the Interaction of Leukocytes to Endothelial Cells and Thereby Regulating Leukocyte/Endothelial Cell-Mediated Responses It has also been discovered, as shown by the teachings herein, that the polypeptides of the present invention have the capacity of inhibit the interaction of Mac-1 receptor-bearing leukocytes to endothelial cells, and thereby inhibit the consequential leukocyte/endothelial-cell mediated responses. Those responses are not limited to inflammation but include leukocyte recruitment, leukocyte adhesion and extravasation, he atopoiesis, antigen presentation, angiogenesis, syncytial formation and he ostasis.

Thus, the present invention provides for a method of inhibiting Mac-1-mediated interaction of leukocytes with endothelial cells thereby inhibiting the leukocyte/endothelial-cell mediated responses in a patient comprising administering to the patient a therapeutically effective amount of an inhibitor of this invention, namely a polypeptide, in a physiologically tolerable therapeutic composition as described herein.

A therapeutically effective amount of an inhibitor is a predetermined amount calculated to achieve the desired effect, i.e., to inhibit in vivo the interaction of circulating Mac-1 receptor-bearing leukocytes with endothelial cells, and thereby reduce the leukocyte/endothelial cell-mediated responses of leukocyte recruitment, leukocyte adhesion and extravasation, hematopoiesis, antigen presentation, angiogenesis, and syncytial formation and the like. The method can be practiced on a patient during an episode of inflammation to reduce the consequential effects of leukocyte interaction with endothelial cells following the injury-induced expression of endothelial cell inflammatory mediators including cytokines, leukotrienes, thromoboxines, and the like. The method can also be practiced on a patient as a prophylactic to inhibit future or anticipated leukocyte/endothelial cell-mediated events by administration of the therapeutic composition prior to the occurrence of a vascular injury.

Mac-1 receptor-bearing leukocyte/endothelial cell-mediated responses occur as a result of the interaction of Mac-1 to an endothelial cell surface ligand. This interaction is induced by local vascular events. Local injury to vasculature and/or the presence of lipopolysaccharide, interleukin-1 (IL-1) , tumor necrosis factor (TNF) and gamma interferon induces the release of cytokines including IL-1, interleukin-6 (IL-6) , colony stimulating factors

(CSFs) , and various leukocyte chemotactic factors such as monocyte-derived neutrophil chemotactic factor/interleukin-8. These factors together induce lymphocyte activation, local and systemic inflammation acute phase responses, hematopoiesis, and leukocyte recruitment and activation.

IL-1 induces the expression of adhesion molecules on the surface of endothelial cells. Such adhesion structures include endothelial leukocyte cell adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2) . Both ELAM-1 and ICAM-1 are ligands for circulating leukocytes which thus promote the interaction of leukocytes with the endothelium. Following the adhesion events, IL-1 then elicits leukocyte extravasation into the underlying tissues by disruption of the integrity of the vessel wall, the result of which is local tissue injury.

In addition to the effects of endothelial cell-produced cytokines resulting in leukocyte recruitment and extravasation, the cytokines induce leukocyte activation which results in the expression and secretion of IL-1, TNF, gamma interferon and growth factors including fibroblast growth factor and transforming growth factor. These additional circulating factors heighten the endothelial cell-mediated secretion of factors which further induce leukocyte interaction with the endothelium resulting in a cascade phenomenon of more factors released and more leukocytes attracted and attaching to the endothelium. Subsequent functional responses resulting from the cascade event include leukocyte recruitment, vasodilation, leukocyte adhesion and extravasation, syncytial formation of leukocytes and endothelial cells, hematopoiesis, angiogenesis, antigen presentation, and thrombosis. Thus, the effects of leukocyte interaction with endothelial cells mediate not only inflammation but also regulate immune functions and hemostasis. The present invention contemplates a method for inhibiting the interaction of leukocytes bearing Mac-1 receptors with endothelial cells through the use of Factor X-derived synthetic polypeptides which block the Mac-1 receptor on the leukocytes thus preventing the interaction of Mac-1 receptor with its corresponding endothelial cell ligand. The result of the inhibition of the leukocyte/endothelial cell interaction thereby inhibits the leukocyte/endothelial cell-mediated responses described herein that are distinct from therapeutic treatment leukocyte/endothelial cell-mediated inflammation.

A therapeutically effective amount of a polypeptide of this invention is typically an amount of polypeptide such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma (intravascular) concentration of from about 0.05 micromolar (urn) to about 1000 uM, preferably from about 0.1 uM to about 500 uM, and more preferably from about 1 uM to about 250 uM. Additional teachings of dosages for effective inhibition can be found in the data described in the Examples.

A therapeutically effective level of inhibition of interaction of Mac-1 receptor-bearing leukocytes with endothelial cells in a patient indicative of the efficacy of the inhibition therapy can be readily determine by a variey of routine clinical analyses to measure signs of leukocyte/endothelial cell-mediated responses. The modes of administration of the therapeutic composition along with the forms of the compositions and regimes for their administration are the same as those described for inhibiting the binding of Factor X to Mac-1.

F. Diagnostic Systems

The present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence of Factor X, or a Factor X polypeptide of this invention in a fluid sample suspected to contain the protein or polypeptide according to the diagnostic methods described herein. A diagnostic system includes, in an amount sufficient for at least one assay, a subject polypeptide and/or a subject antibody or monoclonal antibody of the present invention, as a separately packaged reagent.

In one embodiment, the diagnostic system is useful for assaying for the presence of a polypeptide or anti-polypeptide antibody in a body fluid sample such as for monitoring the fate of therapeutically administered polypeptide or anti-polypeptide antibody. Instructions for use of the packaged reagent are also typically included.

As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits a polypeptide, polyclonal antibody or monoclonal antibody of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide or antibody or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide have been operatively affixed, i.e., linked so as to be capable of being immunologically bound by an antibody.

"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.

In one embodiment, a diagnostic system for assaying for the presence of or to quantitate a serine protease present in a sample, such as blood, plasma or serum, comprises a package containing at least one antibody of this invention, which is immunoreactive with Factor X. The system can additionally contain, typically in a separate package, a polypeptide of this invention having a sequence immunoreactive with the antibody included in the system. Exemplary diagnostic systems utilizing an Factor X inhibitory polypeptide or antibody of this invention are described in the Examples. A diagnostic system of the present invention can additionally include a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule of the present invention. The word "complex" as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immunoreaction products.

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 expressed protein, polypeptide, or 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 proteins methods and/or systems. The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyanate (FITC) , 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC) , tetramethylrhodamine isothiocyanate (TRITC) , lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis, et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982) , which is incorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-amino-di- (3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS) .

Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Cr represent one class of gamma ray emission- producing radioactive element indicating groups. Particularly preferred is ¹²⁵I. Another group of useful labeling means are those elements such as ¹¹C, ¹⁸F, ¹⁵0 and ¹³N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such ¹¹¹ indium of ³H.

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, Aurameas, 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 or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention. Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably the specific binding agent binds the reagent species when that species 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 "ELISA" format to detect the quantity of an inhibitor of this invention in a vascular fluid sample such as blood, serum, or plasma. "ELISA" 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 quantify the amount of an antigen present in a sample. A description of the ELISA 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, which are all incorporated herein by reference. Thus, in some embodiments, a polypeptide or a antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and polypeptides well known to those skilled in the art, can be used.

Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ) ; agarose; beads of 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 powder, e.g., in lyophilized 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 packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. The term "package" refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene and polycarbonate) , paper, foil and the like capable of holding within fixed limits a diagnostic reagent such as a polypeptide, antibody or monoclonal antibody of the present invention. Thus, for example, a package can be a bottle, vial, plastic and plastic-foil laminated envelope or the like container used to contain a contemplated diagnostic reagent or it can be a microtiter plate well to which microgram quantities of a contemplated diagnostic reagent have been operatively affixed, i.e. , linked so as to be capable of being immunologically bound by an antibody or polypeptide to be detected.

F. Assay Methods

The present invention contemplates various assay methods for determining the presence, and preferably amount, of an antigen such as Factor X, a polypeptide or an antibody of the present invention in a fluid sample using a polypeptide, polyclonal antibody or monoclonal antibody of this invention as an immunochemical reagent to form an immunoreaction product whose amount relates, either directly or indirectly, to the amount of the antigen in the sample. This embodiment is particularly useful to monitor the fate of therapeutically administered polypeptides or antibodies as described in the therapeutic methods herein.

Those skilled in the art will understand that there are numerous well known clinical diagnostic chemistry procedures in which an immunochemical reagent of this invention can be used to form an immunoreaction product whose amount relates to the amount of an antigen to be measured that is present in a body sample. Thus, while exemplary assay methods are described herein, the invention is not so limited.

Various heterogenous and homogeneous protocols, either competitive or noncompetitive, can be employed in performing an assay method of this invention. In one embodiment for detecting the presence of

Factor X or a polypeptide of this invention in a fluid sample, the assay method comprises the steps of: (a) Forming an immunoreaction admixture by admixing a fluid sample with an anti-polypeptide antibody of the present invention, preferably a monoclonal antibody, of the present invention.

Insofar as immunoassay depends on the specific reactivity of antigen and antibody, the selection of antibody depends on the species of polypeptide to be detected. Thus, for example, if the assay is designed to detect Factor X, an antibody produced using any of the Factor X-derived polypeptides is admixed with the sample.

Similarly, where the fluid sample contains a polypeptide, an anti-polypeptide antibody immunospecific for the polypeptide is admixed with the fluid sample to form the immunoreaction admixture.

In a related embodiment for detecting the presence of a anti-polypeptide antibody in a fluid sample, the fluid sample is admixed with a polypeptide immunoreactive with the anti-polypeptide antibody rather than with an antibody to form the immunoreaction admixture.

The fluid sample can be a vascular fluid such as blood, plasma or serum, or other body fluid, and is preferably provided as a known amount of blood, or a blood derived product.

Preferably, the amount of antibody or polypeptide as immunochemical reagent that is admixed is known. Further preferred are embodiments where the antibody is labeled, i.e., operatively linked to an indicating means such as an enzyme, radionuclide and the like.

In preferred embodiments, the immunochemical reagent is present as part of a solid support, i.e., operatively linked to a solid matrix, so that the immunoreaction admixture formed has a solid and a liquid phase and the immunochemical reagent functions as a capture reagent. Further preferred are embodiments wherein the amount of polypeptide present in the immunoreaction admixture is an amount sufficient to form an excess of epitopes relative to the number of antibody combining sites present in the immunoreaction admixture capable of immunoreacting with those epitopes. (b) The immunoreaction admixture is then maintained under biological assay conditions for a predetermined time period such as about 10 minutes to about 16-20 hours at a temperature of about 4 degrees C to about 45 degrees C that, such time being sufficient for the inhibitor antigen present in the sample to immunoreact with (immunologically bind) the immunochemical reagent to form an antigen-containing immunoreaction product (immunocomplex) containing the target antigen immunoreacted with the immunochemical reagent. In embodiments where the immunochemical reagent is in the solid phase, the immunocomplex formed is also present in the solid phase.

Biological assay conditions are those that maintain the biological activity of the immunochemical reagents of this invention and the antigen sought to be assayed. Those conditions include a temperature range of about 4 degrees C to about 45 degrees C, a pH value range of about 5 to about 9 and an ionic strength varying from that of distilled water to that of about one molar sodium chloride. Methods for optimizing such conditions are well known in the art.

(c) The amount of antigen-containing immunoreaction product that formed in step (b) is determined, thereby determining the amount of preselected antigen present in the sample.

Determining the amount of the antigen-containing immunoreaction product, either directly or indirectly, can be accomplished by assay techniques well known in the art, and typically depend on the type of indicating means used.

In preferred competitive assay methods, the amount of product determined in step (c) is related to the amount of immunoreaction product similarly formed and determined using a control sample in place of the vascular fluid sample, wherein the control sample contains a known amount of a subject polypeptide or anti-polypeptide antibody from which a standard curve is determined.

Exemplary of the contemplated diagnostic assay, wherein a polypeptide immunoreactive with the antibody reagent is operatively linked to a solid matrix is the ELISA described in the Examples.

Also contemplated are immunological assays capable of detecting the presence of immunoreaction product formation without the use of a label. Such ethods employ a "detection means", which means are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel polypeptides, methods and systems. Exemplary detection means include methods known as biosensors and include biosensing methods based on detecting changes in the reflectivity of a surface, changes in the absorption of an evanescent wave by optical fibers or changes in the propagation of surface acoustical waves.

Thus, in one embodiment, the invention contemplates a method for detecting the presence of a Factor X antigen, such as Factor X or a Factor X- derived polypeptide of this invention in a fluid sample comprising the steps of:

(a) forming an immunoreaction admixture by admixing the fluid sample with an anti-Factor X polypeptide antibody of this invention comprising antibody molecules that immunoreact with (i) Factor X, and with (ii) a polypeptide having an amino acid residue sequence represented by a formula selected from the group consisting of (1:366-373), (1:423- 430), and (1:238-246); but do not immunoreact with the polypeptide having an amino acid residue sequence represented by the formula (1:174-201);

(b) maintaining the immunoreaction admixture for a time period sufficient to form a Factor X antigen- containing immunoreaction product; and (c) detecting the presence of immunoreaction product formed in step (b) and thereby the presence of Factor X antigen in the fluid sample.

The present invention also contemplates a diagnostic method for detecting the presence in a fluid sample of a therapeutically administered anti- polypeptide antibody that immunoreacts with Factor X and with a polypeptide of this invention comprising the steps of:

(a) forming an immunoreaction admixture by admixing a fluid sample with a polypeptide of the present invention that immunoreacts with the anti- peptide antibody;

(b) maintaining said immunoreaction admixture for a time period sufficient to form an immunoreaction product containing said polypeptide; and

(c) determining the presence of the immunoreaction product formed in step (b) , and thereby the presence of said antibody.

Other permutations on the above diagnostic methods using the polypeptides and antibodies of the present are readily apparent to one skilled in the immunological arts, and are also contemplated.

Due to the ability of Factor X to bind Mac-1, an additional assay method is contemplated for detecting the presence of a Factor X polypeptide in a liquid (aqueous) sample, and comprises the steps of:

(a) admixing a sample of activated cells having Mac-1 receptors with (i) a predetermined amount of said liquid sample containing said polypeptide having an amino acid residue sequence that is from 8 to 50 residues in length and includes a sequence represented by a formula shown in a SEQ ID NO selected from the group consisting of (1:366-373), (1:423-430) and (1:238-246) , and (ii) a predetermined amount of labelled Factor X to form a competition reaction admixture;

(b) maintaining said competition reaction admixture for a predetermined time period sufficient for said polypeptide present in said admixture to bind to said Mac-1 receptors and form a Mac-1 receptor:polypeptide complex and form said labelled Factor X to bind to said Mac-1 receptors to form a labelled Mac-1 receptor:Factor X complex; and

(c) assaying for the amount of labelled Mac-1 receptor:Factor X complex formed in step (b) thereby detecting the amount of a Factor X in said sample.

Preferably, the activated cells are monocytes, and more preferably are THP-1 cells.

Preferably, the polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of (1:363-375), (18:1-9), (1:366-373), (1:417-431), (19:1-9), (1:422-430), (1:423-430), (1:237-262), (7:1-10), (7:2-11), (7:1- 11), (8:1-18), (1:238-246) and (1:238-255). The above Mac-1-dependent assay method for detecting a polypeptide of this invention in a fluid sample is described in detail in the Examples.

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 limiting the invention. Variations and equivalents, now known or later developed, which would be within the understanding and technical competence of one skilled in this art are to be considered as falling within the scope of the invention.

1. Synthesis of Polypeptides

Polypeptides were derived from the amino acid residue sequence of mature human Factor X zymogen protein of 448 amino acid residues in length (SEQ ID NO 1) which is composed of a light and a heavy chain. See, Leytus et al. , Proc. Natl. Acad. Sci. , USA. 81:3699-3702 (1984) and Fung et al., Proc. Natl. Acad. Sci.. USA. 82:3591-3595 (1985). The light chain begins and ends at amino acid residue position 1 and 139, respectively. Putative cleavage sites for the removal of a tripeptide linking the light chain to the heavy chain are located between amino acid residue positions 139 and 140 and between 142 and 143. Cleavage at these positions results in the formation of a two-chain Factor X held together by a single disulfide bond between the light chain cysteine residue at position 132 and the heavy chain cysteine residue at position 302. The heavy chain of Factor X begins and ends at amino acid residue position 143 and 448, respectively. Activation of Factor X (Xa) is accomplished by cleavage of a specific arginine- isoleucine peptide bond between amino acid residue positions 194 and 195. This process is activated through either the intrinsic or extrinsic coagulation pathways as described before in which a 11 kd activation peptide having 52 amino acid residues is lost. The resultant activated Factor X (Xa) is 254 amino acid residues in length. These 254 amino acids are also referred to herein as the serine protease of catalytic domain of Factor X. Partially overlapping polypeptides derived from the sequence of human Factor X and polypeptides having deletions, substitutions or additions of particular amino acid residues were synthesized using the classical solid-phase technique described by Merrifield, Adv. Enzv ol.. 32:221-296 (1969) as adapted for use with a model 430 automated peptide synthesizer (Applied Biosyste s, Foster City, CA) . Prepared polypeptide resins were cleaved by hydrogen fluoride, extracted and analyzed for purity by high pressure liquid chromatography (HPLC) using a reverse-phase C18 column manufactured by Waters Associates, Milford, MA. After purification, the synthetic polypeptides were separately dissolved in water at neutral pH at a stock concentration of 5 to 10 mM. In some polypeptides, glycine residues were added at either the amino- or carboxy-terminal end of the polypeptide or at both ends to increase polypeptide stability. Variant polypeptides were also synthesized with substitutions and/or deletions. The domains of Factor X from which the polypeptides were derived are also indicated in the table.

The synthesized polypeptides are listed in Table 1 with their designated SEQ ID NO. The residue numbers corresponding to specific regions in the native Factor X protein are also shown for each polypeptide. Polypeptides having a sequence identical to a portion of Factor X are shown as SEQ ID NO 1. These polypeptides are referred to by their corresponding amino acid residue position in native Factor X protein sequence shown in SEQ ID NO 1. Two conventions are used in the specification in referring to these polypeptides. For example, polypeptide 130-139 that exactly corresponds to the amino acid residue position in Factor X light chain is referred to by SEQ ID NO 1:130-139 or polypeptide 1:130-139. Note that the latter convention lacks the words "SEQ ID NO" but does have the sequence listing number of native Factor X followed by a colon and the corresponding amino acid residue positions in Factor X. In the Examples, polypeptides are referred to with the latter convention.

Polypeptides having additional amino acid residues, deletions and/or substitutions from the native Factor X sequence are listed with separate SEQ ID NO. These variant Factor X polypeptides are also designated by the region they share in common with Factor X followed by an asterisk which denotes that the polypeptide sequence deviates in some way from the native Factor X sequence. For example, polypeptide 56-60* is shown in SEQ ID NO 2 as it has glycine residues added at the amino- and carboxy-terminal ends of the native Factor X amino acid residue sequence from positions 56-60. For example, variant polypeptide 56-60* is referred to as polypeptide 56-60* (SEQ ID NO 2) . The exact amino acid residue additions, deletions and/or substitutions are shown in the Features section of each SEQ ID NO listing.

Table 1 Factor X Polypeptides and Variants Thereof

Corresponding Amino

₁ 249-260 Heavy Chain

₁ 246-260 Heavy Chain

1 240-262 Heavy Chain

1 242-262 Heavy Chain 1 246-262 Heavy Chain

1 246-264 Heavy Chain

11 238-260* Heavy Chain 1 254-269 Heavy Chain

12 253-269* Heavy Chain 13 253-270* Heavy Chain

14 269-283* Heavy Chain 1 284-303 Heavy Chain

15 305-320* Heavy Chain

16 325-338* Heavy Chain 17 332-344* Heavy Chain

1 363-375 Heavy Chain

18 366-373* Heavy Chain 1 384-394 Heavy Chain 1 404-414 Heavy Chain 1 417-431 Heavy Chain

1 422-430 Heavy Chain

19 423-430* Heavy Chain 1 421-430 Heavy Chain 1 420-430 Heavy Chain 1 419-430 Heavy Chain

* = Deletions and/or Substitutions in Synthetic Polypeptides.

** = Numerical polypeptide designations exclude amino- or carboxy-terminal substituted residues if not Factor X.

2. Preparation of Factor X a. Isolation Human plasma Factor X, a vitamin K-dependent protein, was isolated from one kilogram of Cohn fraction III isolated from human plasma from normal donors as described by Fair et al., J. Clin. Invest. , 64:884-894 (1979). The Cohn fraction III was first extracted in 15 liters of 0.01 M Tris-HCl (Tris[hydroxymethyl]-aminomethane) at pH 9.0, 0.025 M sodium citrate, 0.15 M sodium chloride, 2 mM benzamidine and 15,000 Units of heparin for one hour at 4C (4 degrees Celsius) . All subsequent procedures were also performed at 4C. The solution was clarified by centrifugation at 1500 X g for 20 minutes. Admixed with the resultant supernatant was 1.05 liters of 1 M barium chloride to form a barium citrate precipitate which was subsequently collected by centrifugation.

The resultant pellet was washed twice with water and then resuspended in a solution containing 0.05 M morpholino-ethanesulfonic acid-Tris-HCl at pH 5.85 containing 0.2 M sodium citrate, 0.01 M benzamidine and 3000 Units of heparin to a total volume of three liters. The suspension was maintained overnight. After centrifugation of the suspension at 10,000 X g for 20 minutes, solid ammonium sulfate was then admixed with the resultant supernatant to 40% saturation and the admixture was maintained under mixing conditions for 30 minutes. After centrifugation of the admixture, the resultant supernatant was admixed with ammonium sulfate to 70% saturation and maintained under mixing conditions for 45 minutes. The solution was then centrifuged at

10,000 X g for 20 minutes and the resultant pellet was resuspended in a minimal volume of a buffer containing 0.02 M Tris-NaOH, pH 5.85, 0.1 M EDTA (ethylenediaminetetraacetic acid) and 2 mM benzaminidine and then dialyzed against one liter of the buffer overnight followed by a second dialysis against one liter of 0.02 M Tris-H₃P0₄ at pH 5.85 containing 2 mM benzamidine and 0.02% sodium azide. Purification of Factor X and removal of contaminating proteins was accomplished through three different types of column chromatography as follows. First, the dialyzed solution containing Factor X was then applied to a DEAE cellulose column (Whatman Inc., Clifton, NJ) equilibrated with the final dialysis buffer. Factor X immobilized on the column was eluted over 48 hours with a linear gradient from 0.10 to 0.35 M NaCl in the initial buffer at 55 milliliters (ml)/hour. After concentration and dialysis of the eluted protein peak against one liter of 0.15 M potassium phosphate at pH 6.8 containing 1 mM benzamidine and 0.02% sodium azide, the protein solution was then applied onto a hydroxylapatite- Sephadex G-25 column (Pharmacia, Piscataway, New Jersey) that had been equilibrated with the potassium phosphate buffer described above. Factor X was eluted from the column over 48 hours with a linear gradient from 0.15 to 0.30 M potassium phosphate at pH 6.8 containing 1 mM benzamidine and 0.02% sodium azide at 16 ml/hour. After concentration and dialysis against 0.05 M morpholinoethanesulfonic acid-Tris-HCl at pH 5.85 containing 2 mM benzamidine and 0.02% sodium azide, Factor X was then applied to a benzamidine- Sepharose column equilibrated with the morpholinoethanesulfonic acid dialysis buffer. Immobilized Factor X was eluted over 24 hours using a linear gradient from 0.1 to 0.4 M NaCl at 17 ml/hour. The Factor X-containing peak was then applied to a homoarginine-Sepharose column equilibrated with 0.02 M morpholinoethanesulfonic acid-Tris-HCl at pH 5.85 containing 0.02% sodium azide. Factor X was eluted from this column with a linear gradient from 0.2 to 2 M NaCl in morpholinoethanesulfonic acid-Tris-HCl at pH 5.85 containing 0.02% sodium azide. After concentrating the eluted protein peak, Factor X was then applied to a Sephadex G-100 column (Pharmacia) . The purified Factor X eluted from the Sephadex G-100 column showed a constant specific activity over the protein profile and was concentrated and stored at -70C. Factor X concentration was determined by absorbance at 280 nm using an extinction coefficient of 11.6. The purified Factor X was determined to be devoid of activated Factor X (Xa) as measured in a coagulation assay as described by Miletich et al., J. Biol. Chem.. 253:6908-6919 (1978) . Briefly, Factor Xa was assayed by admixing 75 microliters (ul) of purified Factor X sample, 75 ul of 25 mM calcium chloride, and 75 ul of 400 ug/ml of rabbit brain cephalin. The admixture was maintained at 37C for 30 seconds and the coagulation reaction was initiated by admixture of 75 ul of factors VII- and X- deficient bovine plasma at 37C. A standard curve was constructed by assaying dilutions of the sample with the highest specific activity, b. Characterization Factor X isolated as described above was electrophoresed under non-reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The molecular weight of the electrophoresed purified Factor X was estimated to be 64,000 daltons when compared to molecular weight standards. Under reducing conditions, the Factor X was cleaved into two subunits, a heavy chain of 47 kilodaltons (kd) and a light chain of 17 kd. ^c- Radioactive Labeling of Factor X Purified Factor X was radiolabeled with sodium iodide (Na¹²⁵I) by the Iodogen method as described by Fraker et al. , Biochem. Biophys. Res. Commun.. 80:849-857 (1978) and by manufacturer's instructions (Pierce Chemical Co., Rockford, IL) . Briefly, 33 micrograms (ug) of l,3,4,6-tetrachloro-

₃a,6a-diphenylglycoluril in dichloromethane were dried in a glass tube under nitrogen. Admixed into the glass tube were 200 ug of Factor X in phosphate buffered saline (PBS) at pH 7.2 and 2 mCi of carrier-free Na¹²⁵I (Amersham Corp., Arlington Heights, IL) . After 15 minutes on ice, ¹²⁵I-Factor X was isolated by chromatography on Sephadex G-25 (Pharmacia) . The specific radioactivity of ¹²⁵I-Factor X ranged from 0.3 to 1 uCi/ug protein. The specific functional activity of purified Factor X was 130 units/milligram (mg) of protein and was not significantly decreased by radioiodination.

3. Identification of Factor X Polypeptides that Bind to Mac-1 Integrin Receptor a. Inhibition of Factor X Binding to Mac-1 Receptor by Synthetic Polypeptides (1) Preliminary Screening

Initiation of coagulation on vascular cells is implicated in various immune and inflammatory reactions and contributes to vascular injury and atherogenesis. Niemitz et al. , Nature New Biol. , 232:247 (1971) . Leukocytes, platelets and endothelial cells each interact with proteins through regulated, receptor-mediated assembly. On stimulated monocytes, the Mac-1 receptor (CDllb/CD18) , a leukocyte- restricted member of the integrin gene superfamily, binds with high affinity the coagulation serine zymogen Factor X. Altieri et al. , J. Biol. Chem.. 263:7007-7015 (1988) and Altieri et al. , Proc. Natl. Acad. Sci. , USA, 85:7462-7466 (1988). To determine the Mac-1 receptor-binding site or sites within the catalytic domain on the native Factor X zymogen protein, the partially overlapping synthetic polypeptides prepared in Example 1 were separately evaluated in a quantitative receptor:ligand binding assay.

In this assay, each polypeptide was initially tested for its ability to competitively inhibit the binding of ¹²⁵I-labeled Factor X to Mac-1 integrin receptors expressed on the human monocytic cell line, THP-1 (ATCC Accession No. TIB 202, American Tissue Culture Collection, Rockville, MD) and the inhibition of binding was measured as described below. THP-1 cells were maintained in RPMI 1640 (Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine serum (Irvine Scientific) , 25 mM Hepes, 1 mM L-glutamine (Irvine Scientific) , 100 ug/ml gentamycin (Geramycin; Schering Plough, Kenilworth, NJ) , and 10 uM beta- mercaptoethanol (Eastman Kodak, Rochester, NY) .

For the polypeptide competition experiments, separate 0.2 ml aliquots of THP-1 cells resuspended at 1.5 X 10⁷ cells/ml in serum-free RPMI 1640 were stimulated with 10 uM of the cytosolic calcium mobilizing peptide formyl-methionyl-leucyl- phenylalanine (N-fMLP, Sigma Chemical Co., St. Louis, MO) in the presence of 2.5 mM CaCl₂ and simultaneously admixed with 15 nM ¹²⁵I-Factor X prepared in Example 2c and 0.5 mM doses of various Factor X synthetic polypeptides prepared in Example 1 to form separate competition reaction admixtures. The admixtures were maintained for 20 minutes at 22C. The reaction was terminated by centrifugation at 12,000 X g for 5 minutes at room temperature through a mixture of silicone oils (Dow Corning, New Bedford, MA) . The total cell-associated radioactivity was determined by gamma counter detection. Specific or net cell-associated binding of ¹²⁵I-Factor X was then calculated by subtracting the cell-associated radioactivity that resulted in control admixtures maintained in the presence of 100-fold molar excess of unlabeled Factor X from the total radioactivity. The specific or net ¹²⁵I-Factor X bound to N-fMLP- stimulated THP-1 cells in the absence of competing polypeptides was determined to be at a density of

57,000 ± 8,000 molecules/cell from a sample size of 8 experiments. The reduction in specific binding of ¹²⁵I-Factor X to Mac-1 in the presence of admixed polypeptides was then measured and presented as percent of inhibition of net ¹²⁵I-Factor X binding.

The more competitive the polypeptide, the greater the percentage of inhibition. The results of the polypeptide competition experiments are shown below in Table 2 for each polypeptide evaluated. The polypeptides tested are listed in Table 2 according to their SEQ ID NO and the corresponding amino acid residue position relative to native mature Factor X. The amino acid residue sequence of the polypeptides listed in Table 2 are presented in the Sequence Listing.

Table 2 Percent of Inhibition of Factor X Binding

The three polypeptides, 1:237-262, 1:363-375 and 1:417-431, inhibited the binding of Factor X to Mac-1 receptors on THP-1 cells at greater than 70%. The inhibition was mediated through the binding of the polypeptides to the ligand-binding sites on Mac-1 and not through any interaction with Factor X itself. The three inhibitory polypeptides comprised non-contiguous linear sequences in the heavy chain (catalytic) domain of Factor X. The EGF-like polypeptide 1:82-89 and heavy chain polypeptides 1:305-320 and 1:332-344 did not produce titratable dose-dependent inhibition of ¹²⁵I-Factor X binding to THP-1 cells. The apparent increase in ¹²⁵I-Factor X binding in the presence of heavy chain polypeptides 1:253-270, 1:384-394 and 1:404-414 was not observed when the polypeptides were evaluated at lower doses of 0.1 mM, thus, reflecting self-aggregation of the ligand. Thus, only the three polypeptides, 1:237-262, 1:363-375 and 1:417-431, were determined to competitively inhibit the binding of Factor X to Mac-1 receptors on the monocyte-like THP-1 cells.

Consistent with the interaction of the inhibitory Factor X-derived polypeptides with Mac-1 rather than with Factor X itself, none of the three inhibitory polypeptides affected the mechanism of Factor X activation or the proteolytic activity of the activated form of Factor X, Xa, as described by Fair et al., J. Biol. Chem., 264:11035-11043 (1989). In that study, Fair et al. mapped the surface regions of Factor X that mediated the association of Factor X with activator molecules of Factor X. The activators were shown to induce the cleavage of inactive Factor X to Xa. Components required for activation of Factor X included activated factor VII (Vila) and tissue factor in the extrinsic activation pathway, activated factors IX and VIII in the intrinsic activation pathway and lastly, an enzyme from Russell•s viper venom (RW-X) . The extrinsic and intrinsic pathways are membrane- mediated events whereas RW-X is a soluble non- membrane-mediated event.

Fair et al. identified the activation recognition sites by measuring the effects of Factor X-derived synthetic polypeptides on the rates of Factor X activation in amidolytic assays. Three non-contiguous polypeptides derived from the amino acid residue sequence of the heavy chain blocked the binding sites of the activator molecules interfering in the orientation of the activator on the surface of Factor X. This misalignment of the activator with Factor X did not allow for the cleavage of Factor X to form Xa. Although many of the polypeptides tested by Fair et al. were identical or partially homologous to the polypeptides used in the instant invention, the former were used to map the sites on Factor X to which activators of the extrinsic, intrinsic or RW-X pathways bind and induce the conversion of the Mac-1-bound inactive Factor X to activated Factor X. In contrast, the polypeptides in the instant invention were used in the inhibition of Factor X binding assays described above to map the Mac-1 receptor-binding sites on Factor X. Three separate regions of the Factor X heavy chain were defined by this approach to comprise Mac-1 receptor-binding sites. These three sites coordinately mediated the binding of Factor X to Mac-1 receptors expressed on monocytes as described below in Example 3a(2) and 3b. This monocyte membrane-mediated event is distinct from those membrane-mediated events in the extrinsic and intrinsic pathways. Binding of Factor X to Mac-1 receptors on monocytes and its subsequent cleavage to Xa, thus, represents a fourth and alternative pathway for the activation of Factor X which is independent from the classical extrinsic, intrinsic and RW-X activation pathways.

Fair et al. showed that the most effective polypeptides at inhibiting the rate of Factor X activation in the classical activation pathways were polypeptides 1:267-283, 1:284-303 and 1:417-431, listed in order of decreasing effectiveness. The polypeptides 1:267-283 and 1:284-303 completely inhibited the formation of Factor Xa in both the extrinsic and intrinsic pathways. In the RW-X system, polypeptide 1:267-283 completely inhibited Factor Xa formation while polypeptide 1:284-303 resulted in greater than 65% inhibition. The polypeptide 1:417-431 only minimally interfered with Factor X activation mediated through the classic extrinsic coagulation pathway and did not inhibit Factor X activation through either the intrinsic pathway or with RW-X.

In the instant invention, the Fair polypeptides 1:267-283 and 1:284-303 failed to inhibit the binding of Factor X to Mac-1. Thus, those corresponding regions on Factor X only function in the activation of Factor X as Fair et al. demonstrated. Only Fair's polypeptide 1:17-431 was, in the instant invention, a potent inhibitor of the binding of Factor X to Mac-1. Given that polypeptide 1:417-431 only minimally inhibited the activation of Factor X in Fair's systems, the corresponding region in Factor X, thus, is primarily involved in regulating the binding of Factor X to Mac-1 and not its subsequent activation. The regions on Factor X corresponding to polypeptide

1:237-262 and 1:363-375 have been shown in the instant invention to promote the binding of Factor X to Mac-1. Since identical polypeptides do not inhibit Factor X activation, these two regions, 1:237-262 and 1:363-375, in addition to the region corresponding to polypeptide 1:417-431, on Factor X only mediate the binding of Factor X to Mac-1 function shown in the present invention.

In sum, the regions corresponding to polypeptides 1:237-262, 1:363-375 and 1:417-431 respectively comprise the three recognition sites designated 3, 1 and 2 of the Factor X catalytic domain. Variant polypeptides synthesized with a shorter length and/or amino acid residue permutations were then evaluated as described below in order to define the minimum inhibitory sequence for each of the three recognition sites. This analysis described in (2) below thus allowed for the determination of the minimum sequence of amino acid residues in each site which would promote the binding of Factor X to Mac-1.

(2) Secondary Screening to Define the

Minimum Amino Acid Residue Sequence of the Mac-1 Recognition Sites 1. 2 and 3 of Factor X To determine the minimum sequence of the three recognition sites of Factor X which promoted the binding of Factor X to Mac-1, shorter and variant polypeptides were synthesized as described in Example 1 and used in inhibition of Factor X binding assays as described in Example 3a(1) above. The polypeptides and the concentrations at which they were evaluated are shown in Table 3 below. The polypeptides are listed in Table 3 according to their SEQ ID NO and the corresponding amino acid residue position relative to native mature Factor X. The amino acid residue sequence of each polypeptide is shown in the Sequence Listing according to the designated SEQ ID NO. The table is divided into the three groups corresponding to the three recognition sites described in herein.

* Deleted amino acid residue.

** Numerical polypeptide designations exclude a ino- or carboxy-terminal substituted residues of native

Factor X.

1 Added carboxy-terminal glycine residue.

2 Added amino-terminal glycine residue.

3 Added amino- and carboxy-terminal glycine residue. 4 Three amino acid residue substitutions of positions 248, 254 and 255.

5 One amino acid residue substitution at position 248 three deletions at amino acid residue positions 245-247.

6 One amino acid residue substitution at position 248.

7 One amino acid residue substitution at position 248 and three deletions at amino acid residue positions 245-247.

8 One amino acid residue deletion at position 257 and a tryptophan residue substitution for two valines at positions 267 and 268.

The results of these secondary polypeptide competition experiments are shown in Table 3 as percent of inhibition of specific ¹²⁵I-Factor X binding to Mac-1 on THP-1 cells for each polypeptide evaluated. The results are discussed below according to the three recognition sites on Factor X which mediate the interaction of Factor X with Mac-1 on THP-1 cells.

Recognition Site 1:

Polypeptide 1:363-375, at a concentration of 500 uM, inhibited 78% of ¹²⁵I-Factor X from binding to Mac-1 receptors on THP-1 cells. The smaller polypeptide, 366-373* (SEQ ID NO 18) having an added carboxy-terminal glycine residue, comparably inhibited the binding of Factor X to Mac-1 but did so at 50 fold less in concentration. Thus, the minimum amino acid residue sequence which comprises recognition site 1 on Factor X corresponds to the region defined by the polypeptide 1:366-373. The preferred polypeptide amino acid residue sequence comprising recognition site 1 corresponds to the region of Factor X defined by the polypeptide 1:366-373. Recognition Site 2

Two shorter polypeptides corresponding to recognition site 2 were effective at inhibiting the binding of Factor X to Mac-1. Polypeptide 1:422-430, at a concentration of 10 uM, inhibited 70% of the binding while polypeptide 423-430* (SEQ ID NO 19) having an additional amino-terminal glycine residue inhibited 60% of Factor X binding at a polypeptide concentration of 100 uM. Thus, both polypeptides were better inhibitors of Factor X binding than the larger polypeptide 1:417-431 corresponding to recognition site 2 which inhibited 75% of Factor X binding but did so at a high polypeptide concentration of 500 uM. Three other polypeptides, 1:421-430, 1:420-430 and

1:419-430, were not effective at inhibiting Factor X binding.

Thus, the minimum amino acid residue sequence required for maximally inhibiting Factor X binding to Mac-1 corresponds to the region on Factor X defined by the polypeptide 1:422-430 where the native isoleucine residue in position 422 is preferred over the substituted glycine residue. Since polypeptides of intermediate lengths between the minimum length and the maximum length were shown to lack inhibitory activity, they thus do not contribute to the second Mac-1 recognition site on Factor X. The preferred polypeptide amino acid residue sequence comprising recognition site 2 corresponds to the region of Factor X defined by the polypeptide 1:423-430. Recognition Site 3:

The only polypeptides considered as significant inhibitors of Factor X binding to Mac-1 were 238-246* (SEQ ID NO 7) and 238-253* (SEQ ID NO 8) where 238-246* (SEQ ID NO 7) was the most effective inhibitor at concentration of 10 uM compared to 238-253* (SEQ ID NO 8) at 250 uM. The larger polypeptide 1:237-262 also inhibited the binding of Factor X to Mac-1 but at a concentration of 500 uM as was also shown in Example 3a(1). No polypeptides intermediate in length between 1:237-262 and 238-246* (SEQ ID NO 7) and having variant or added amino acid residues were effective at inhibiting Factor X binding. Thus, the preferred polypeptide amino acid residue sequence comprising recognition site 3 corresponds to the region of Factor X defined by the polypeptide 1:238-246.

(3) Dose Response Curves for Inhibition of Factor X Binding to Mac-1 Receptors by the Minimum Recognition Site Factor X

Polypeptides

To determine the concentration of polypeptides at which inhibition of Factor X binding to Mac-1 receptors was at its maximum, inhibition assays were performed as described in Example 3(a)l with the exception that the concentration of the polypeptides, 366-373* (SEQ ID NO 18), 1:422-430 and 238-246* (SEQ ID NO 7) , was varied over the range of 0.1 uM to 10 uM in separate assays. Specific binding of ¹²⁵I-Factor X was calculated as described in Example 3a(1) . The data is plotted as percent of control ²⁵I-Factor X bound in the absence of admixed polypeptides against increasing concentration of polypeptide from 0.1 X 10^"6 M up to 10 X 10^"6 M. The results are shown in Figure 1A. The dose response curves for the polypeptides, 366-373* (SEQ ID NO 18) , 1:422-430 and 238-246* (SEQ ID NO 7), are respectively indicated lines with open circles, lines with open squares and lines with open triangles. Each of the three polypeptides inhibited ¹²⁵I-Factor X binding to Mac-1 on THP-1 cells in a concentration-dependent fashion where the half-maximal (IC₅₀) concentration ranged between 1 to 5 uM.

In addition, Lineweaver-Burke analysis of ¹²⁵I-Factor X binding isotherms in the presence of the three inhibitory polypeptide suggests that the three recognition sites function in a preferred sequential order of addition to govern a correct Factor X alignment on Mac-1. The Lineweaver-Burke analysis revealed that polypeptide 1:422-430 inhibited in a characteristic competitive fashion (control, r=0.99, y'=4.32; polypeptide 1:422-430, r=0.99, y'=4.8) whereas the other two polypeptides produced non- competitive inhibition profiles (366-373* (SEQ ID NO 18), r=0.97, y' =21.7; 238-246* (SEQ ID NO 7), r=0.99, y'=18) . This data suggest that rather than requiring the three sequences simultaneously, proper Factor X alignment may be facilitated by a preferred sequential order of addition, where the non-competitive Factor X loops participate in secondary interactions and cooperatively stabilize receptor-Factor X ligand docking. b. Inhibition of Monocyte Procoagulant Activity Monocyte procoagulant activity was evaluated to determine the effects of the preferred synthetic polypeptides comprising the three recognition sites described in Example 3a on the production and activity of Factor Xa on stimulated monocytic THP-1 cells. Factor X binding to Mac-1 receptors on monocytes has been shown to result in the local generation of thrombin which results in an increase in monocyte procoagulant activity. Altieri et al. , Proc. Natl. Acad. Sci. , USA, 85:7462-7466 (1988). In this assay, in the presence of polypeptides which inhibit the binding of Factor X to Mac-l, Factor X will not bind and thus, the subsequent activation of Factor X will not occur. The absence of activated Factor X will result in a decrease of the downstream effect of coagulant activity (clot formation) as measured by the assay if maintained in the presence of inhibitory polypeptides.

To test this hypothesis, serum-free suspensions of THP-1 cells were first prepared as described in Example 3a(l). Separate aliquots of the prepared cells at 1.5 X 10⁷ cells/ml were then admixed with 100 uM ADP (Sigma) 2.5 mM CaCl₂, 15.1 nM Factor X prepared in Example 2, and 500 uM final concentration of each of the polypeptides, 366-373* (SEQ ID NO 18), 1:422-430 and 238-246* (SEQ ID NO 7) to form procoagulant admixtures. Control experiments were performed in the presence of a scrambled polypeptide. At 5, 20 and 30 minutes after the admixtures were made, 0.1 ml of the reaction admixture was transferred to glass tubes containing 0.1 ml factor VII- and Factor X-deficient bovine plasma (Sigma) and maintained for 1 minute at 37C. The reaction was initiated with 0.1 ml of 0.025 M CaCl₂ and the clotting time was read. Clotting times were measured from the point of addition of plasma to the point of formation of visible clots and were determined in triplicate. A standard curve with serial concentrations from 10-300 ng/ml of Factor Xa was constructed convert clotting times to nanograms (ng) of Factor Xa coagulant activity per ml. The results of this assay at the 30 minute time point are shown below in Table 4. Factor Xa generation, as measured by Factor X coagulant activity described above, was inhibited in the presence of each of the three polypeptides, 366-373* (SEQ ID NO 18) , 1:422-430 and 238-246* (SEQ ID NO 7), as compared to the Factor Xa formation in the presence of a scrambled nonspecific polypeptide. The inhibition of procoagulant activity was also time-dependent. The data are presented as mean ± standard error of the mean (SEM) of three independent experiments. In the presence of each of the three inhibitory polypeptides, Factor Xa formation in ng/ l after a 5 and 20 minute maintenance period was respectively reduced to 14.4 ± 2.1 and 31.8 ± 11.5 (polypeptide 366-373* (SEQ ID NO 18), 16.2 ± 5.7 and 44.3 ± 17 (polypeptide 1:422-430), and 12 ± 0.6 and 30.6 ± 10.5 (polypeptide 238-246* (SEQ ID NO 7) . In the presence of the control scramble polypeptide after the same maintenance periods, Factor X generation was 35.7 ± 2.7 and 177.1 ± 10 ng/ml.

Table 4 Effects of Synthetic Factor X Polypeptides on Monocyte Procoagulant Activity

Polypeptide SEQ Monocyte Procoagulant

ID NO Activity

(ng/ml Xa formed)

346.8 ± 27

95.8 ± 21.8 112 ± 36

118.4 ± 67

The relevance of these observations is that the three synthetic peptidyl analogues comprising the Mac-1 recognition sites on Factor X have the capacity to interrupt generation of thrombin on the surface of vascular cells such as monocytes, thereby preventing the pleiotropic consequences that include chemotaxis, mitogenesis, platelet and leukocyte adhesion of the endothelium and monocyte deposition of insoluble fibrin. Thus, antagonists based on peptidyl analogues or more advanced derivatives may beneficially intervene in related forms of vascular injury by blocking the binding of Factor X to Mac-1 without interfering with physiologic hemostatic mechanisms or leukocyte adhesions reactions. c. Determination of the Localization of the

Mac-1 Recognition Sites in the Catalytic Domain of Factor X

The three polypeptides, 366-373* (SEQ ID NO 18), 1:422-430 and 238-246* (SEQ ID NO 7), were topographically mapped by computational modeling to predicted surface loops of Factor X based on crystallographic data for the homologous zymogens such as chymotrypsinogen and trypsinogen. See, Freer et al., Biochem. , 9:1997-2009 (1970) and Marquart et al., Acta Cristallogr. Sect. B, 39:480 (1983). The energy-minimized structure of the catalytic domain of Factor X indicated that the three peptidyl loci do not cluster in a spatially contiguous region in the molecule to form a single structure. They, instead, are found in three distinct surface loops that surround an unobstructed catalytically active site. The amino-terminus of the heavy chain is located just above polypeptide 366-373* (SEQ ID NO 18) , the substrate binding groove is approximately vertical and to the right of the catalytic triad and the carboxy- terminus of the model is the carboxy-terminus of polypeptide 1:422-430. This structural organization is consistent with a three-dimensionally coordinated tripartite Factor X ligand recognition macromotif for Mac-1. 4. Determination of Specificity of Synthetic Polypeptides for Surface-Expressed gC Giycoprotein on Herpes Simplex Virus fHSV)-Infected Endothelium a. Inhibition of Factor X Binding to gC

Giycoprotein on HSV-Infected Endothelium by Synthetic Polypeptides

Endothelial cells which have been infected with herpes simplex virus (HSV) express the viral giycoprotein C (gC) on their cells surface. The gC giycoprotein has been shown to bind Factor X avidly (Etingin et al. , Cell, 61:657-662 (1990). Using an in vitro model of arterial injury that mimics one of the earliest described pathogenic events in some forms of atherosclerosis, the three peptidyl recognition sites on Factor X were evaluated to determine if the same recognition sites mediated the binding of Factor X to gC.

For the dose-response curve inhibition assay, human Factor X was isolated and radiolabeled as described in Example 2. Human umbilical vein endothelial cells (HUVEC) were obtained from umbilical cords and were subpassaged one to three times prior to use. They were confirmed to be endothelial cells (EC) by i munofluorescent staining with von Willebrand factor antiserum. Etingin et al., supra. Aliquots (0.1 ml) of HUVEC containing 1 X 10⁶ cells were admixed with ¹²⁵I-Factor X at 15 nM, 2.5 mM CaCl₂ and Factor X synthetic polypeptides, 366-373* (SEQ ID NO 18), 1:422-430 and 238-246* (SEQ ID NO 7), prepared in Example 1 and ranging in concentration from 10 to 1000 uM, to form separate competition reaction admixtures. The admixtures were maintained as described in Example 3a(1) . The inhibition of ¹²⁵I-Factor X binding to gC by the synthetic polypeptides was calculated as described in Example 3a(l) .

The results of the inhibition of ¹²⁵I-Factor X binding to gC assays are shown in Figure IB. The data, plotted as described for Figure 1A, shows that the same three peptidyl recognition site sequences that inhibited the binding of Factor X to Mac-1 also inhibited the binding of ¹²⁵I-Factor X to the gC giycoprotein expressed on HSV-infected HUVEC in a concentration-dependent fashion with an IC₅₀ of 60 to 80 uM. Control polypeptides in which the sequence was scrambled did not diminish the ¹²⁵I-Factor X binding to HSV-infected endothelium. These results indicate that although unrelated in primary sequence and overall architecture, the HSV-encoded gC and the leukocyte integrin Mac-1 have evolved through convergent evolution a similar ligand recognition repertoire both including complement proteins of the C3b cascade and Factor X. b. Inhibition of Monocyte Adhesion to HSV-Infected Endothelium by Synthetic

Polypeptides

The effect of the three polypeptides, 366-373* (SEQ ID NO 18), 1:422-430 and 238-246* (SEQ ID NO 7) , on monocyte adhesion to control or HSV-infected endothelium was also evaluated. The assay was performed as described by Etingin et al., supra. Briefly, monocyte cell adhesion was measured using ⁵¹chromium-labeled monocytic THP-1 cells. Monocytes, i.e., mononuclear cells, isolated from peripheral blood, can also be used in these assays. In each experiment, THP-1 cells were used at greater than 10-fold excess over the number of HUVECs. Labeled THP-1 cells at a concentration of 1 X 10⁶ cells/well and 500 uM of each of the above polypeptides were separately admixed to separate monolayers of HUVECs in 24-well plates and maintained at 4C. After 2 hours, the culture medium containing nonadherent THP-1 cells was removed and the wells were gently rinsed twice with culture medium. Sodium hydroxide (0.2%) was then admixed to each well for one hour to disrupt cells prior to counting in a gamma counter. Target cell number was measured in parallel wells. Nonspecific adherence of THP-1 cells to blank wells ranged from l%-4%. The results of those experiments are shown in Table 5. The data is presented as mean ± SEM of three independent experiments.

Table 5 Effects of Synthetic Factor X Polypeptides on Monocyte (THP-1) Adhesion to HSV-infected HUVEC

Polypeptide SEQ Monocyte (THP-1)

ID NO Adhesion to EC (Monocyte bound/EC)

3.3 ± 0.4

1.48 ± 0.16

1.54 ± 0.17

1.46 ± 0.26

All three synthetic polypeptides tested significantly inhibited the adhesion of THP-1 cells to EC cells as compared to control experiments. Control THP-1 cell adhesion to non-infected or HSV-infected endothelium was 1.54 ± 0.29 and 3.4 ± 0.5 THP-1 cells bound/endothelial cell, respectively. THP-1 adhesion to HSV-infected endothelium in the presence of control scrambled versions of each polypeptide was 3.3 ± 0.4 (polypeptide 366-373* (SEQ ID NO 18) ) , 3.2 ± 0.65 (polypeptide 1:422-430) and 3.2 ± 0.3 (polypeptide 238-246* (SEQ ID NO 7)) THP-1 cells bound/endothelial cell. The relevance of these observations is that the three synthetic peptidyl analogues comprising the recognition sites on Factor X which promote the binding to gC expressed on the surface of HSV-infected endothelium have the capacity to interrupt generation of thrombin on the surface of endothelial cells thereby preventing the pleiotropic consequences of thrombin production. Thus, antagonists based on peptidyl analogues or more advanced derivatives may beneficially intervene in related forms of vascular injury by blocking the binding of Factor X to gC resulting in the decreased adhesion of monocytes to HSV-infected endothelium without interfering with physiologic hemostatic mechanisms or leukocyte adhesions reactions. Thus, the zymogen of the coagulation protease cascade Factor X has been shown in this invention to contain a vascular cell binding domain consisting of three non-contiguous recognition sites that binds to leukocyte Mac-1 integrin and viral-infected endothelial gC.

5. Inhibition of Monocyte Adhesion to TNF and IL-1 Stimulated HUVEC by Factor X Synthetic Polypeptides The Factor X polypeptides of this invention as described in the Examples above have been shown to inhibit the binding of Factor X to Mac-1 bearing THP-1 monocytic cells and gC-expressing HUVEC and also inhibit the binding of Mac-1 receptor-bearing THP-1 cells to gC-expressing HUVEC. Not only are endothelial cells responsive to exposure of HSV by the subsequent expression of gC on their cell surface, they are known to produce cytokines, such as tumor necrosis factor, interleukin-1, interleukin-6 and various chemotactic factors, and be acted upon by those cytokines in addition to gamma interferon, granulocyte-colony stimulating factor, granulocyte-macrophage colony-stimulating factor, transforming growth factor beta and fibroblast growth factors. Cytokines have been shown to mediate the complex bi-directional interactions between immunocompetent cells and the vascular endothelium. Cytokines effect endothelial cell function in inflammation, thrombosis, angiogenesis, hematopoiesis, the extravasation of leukocytes and the like. Thus, the regulation of cellular interaction between circulating immunocompetent cells and the vascular endothelium is of great significance. The inhibition of interaction assays described herein were therefore performed to determine whether the inhibitory Factor X synthetic polypeptides would also inhibit the binding of Mac-1 bearing THP-1 monocytic cells to cytokine-activated endothelial cells. The effect of inhibiting the interaction of circulating immunocompetent cells with endothelial cells in vivo would be the mediation of the consequent functional effects such as those listed above.

Human umbilical vein endothelial cells (HUVEC) , commercially available from Clonetics, San Diego, CA, were passaged into 40 gelatin-coated T75 tissue culture flasks (Falcon, Thousand Oaks, CA) and maintained in endotoxin-free RMPI 1640 (Whittaker M.A. Bioproducts, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Sterile Systems, Logan, UT) , 25 mM Hepes [4-(2-hydroxyethyl)-1-peperidineethanesulfonic acid] (Calbiochem Boehring, La Jolla, CA) , 100 ug/ml penicillin-streptomycin-fungizone (Whittaker), 0.5% endothelial cell growth factor (Biomedical Technologies, Stoughton, MA) and 1 mM glutamine (Whittaker) .

For the inhibition of monocyte interaction studies, the HUVEC cultures were first exposed for 4 hours at 37C to either tumor necrosis factor alpha (TNF) (Genzyme Corp. , Cambridge, MA) at a concentration of 100 Units/ml or to interleukin-1 (IL-1) (Genzyme) at a concentration of 25 Units/ml. Following the exposure period, the cells were washed prior to the admixture of selected Factor X polypeptides separately admixed with ⁵¹-Cr-labelled

THP-1 cells. The THP-1 cells, prepared and stimulated with N-fMLP as described in Example 3al) , were first labelled with ⁵¹Cr as described in Example 4b. For the inhibition of interaction assays, increasing concentrations ranging between 1 and 250 uM of selected Factor X polypeptides were separately admixed with approximately 1 X 10⁶ N-fMLP-treated ⁵¹Cr-labelled THP-1 cells. The following polypeptides were examined for their inhibitory activity in the assays: polypeptide 366-373* (SEQ ID NO 18); polypeptide

1:422-430 (SEQ ID NO 1); and polypeptide 238-246* (SEQ ID NO 7) . In some assays, two or more polypeptides were combined together to determine if they would result in additive or synergistic inhibitory effects on the interaction of Mac-1 bearing THP-1 cells with HUVEC.

The resultant polypeptide-THP-1 admixtures were then immediately admixed to monolayers of either TNF- or IL-1-treated HUVEC. After 30 or 45 minutes at 22C, the wells were washed 3 times to remove nonadherent THP-1 cells. The cells left in each well were then solubilized with 20% SDS for counting. The number of labelled-THP-1 cells specifically attached to cytokine-treated HUVEC under the various treatment conditions was then quantitated by measuring the radioactivity in a beta scintillation counter. The total counts obtained from each well was divided by the average of 3-6 counts/cell to obtain the relative number of attached cells in each well. The resultant values were then compared to counts seen in the presence of control scrambled peptides which were then normalized to represent 100% of cell attachment. The data is presented as percent of inhibition compared to the normalized controls. The inhibition of interaction results obtained on TNF- and IL-1-treated HUVEC are respectively shown in Tables 6 and 7.

Table 6 Effects of Synthetic Factor X Polypeptides on Monocyte fTHP-1) Adhesion to TNFα-Stimulated HUVEC

SEQ Polypeptide ID NO

366-373* 18

38-246*

22-430

66-373* + 238-246*

366-373* + 10 10

238-246* + 50 15

422-430 100 42

Control Scrambled

Peptides 100 0

As shown in Table 6, all three synthetic polypeptides exhibited dose-dependent inhibition of the interaction of THP-1 cells to TNF-treated HUVEC. Maximal inhibition of cell was achieved with polypeptide 366-373* at 100 ug/ml followed by polypeptide 238-246* then 422-430. When the first two polypeptides were combined at either a dose of 50 ug/ml or 100 ug/ml of each polypeptide, the inhibitory effect on the interaction of THP-1 cells with HUVEC was diminished. Thus, the effects were not additive nor synergistic and were somewhat competitive resulting in less inhibition than that obtained with each polypeptide when used alone. A similar dose response curve albeit with a enhanced inhibitory effect was obtained when all three polypeptides, each a concentrations of either 10, 50 or 100 ug/ml, were used in combination.

Table 7

Effects of Synthetic Factor X Polypeptides on Monocyte fTHP-1) Adhesion to IL-1-Stimulated HUVEC

422-430 238-246*

366-373* +

238-246*

Control Scrambled Peptides 100

The Factor X polypeptides that exhibited inhibitory activity on the interaction of Mac-1-bearing THP-1 cells with TNF-treated HUVEC showed the same effects on IL-1-treated HUVEC as shown in Table 7. Similar inhibitory dose-response curves were observed over the concentrations of the three individual polypeptides tested. In contrast with the TNF-treated cells, however, when two polypeptides were used in combination at an individual concentration of 100 ug/ml each, the percent of inhibition obtained was equivalent to that seen with polypeptide 366-373* used alone. The effect which was not further inhibited as was seen with TNF-treated cells was nevertheless still not additive nor synergistic.

In summary, the Factor X polypeptides that inhibited the binding of Factor X to Mac-1 on THP-1 cells and also inhibited the Mac-1-mediated binding of THP-1 cells to gC-expressing HUVEC have now been shown to be effective mediators of the interaction between THP-1 monocytic cells and HUVEC. Thus, Factor X polypeptides inhibited the interaction via a Factor X site on Mac-1, the effect of which subsequently blocked the interaction of Mac-1 to cell surface ligands. Intercellular adhesion molecule-1 (ICAM-1) has been previously shown to be expressed on endothelial cells in response to TNF and IL-1 exposure. Thus, Mac-1 receptor-mediated binding to HUVEC may be achieved through a cell surface ligand such as ICAM-1 and inhibited from accomplishing that interaction by Factor X-derived synthetic polypeptides. The regulation of the cellular interaction between circulating immunocompetent cells and the vascular endothelium may therefore be accomplished with the use of antagonists based on peptidyl analogues, such as Factor X-derived synthetic polypeptides, the result of which in vivo would be a mediation of endothelial cell function in inflammation, thrombosis, angiogenesis, hematopoiesis, leukocyte recruitment, leukocyte adhesion and extravasation, antigen presentation, syncytial formation, and the like.

6. Preparation of Polyclonal Antisera to Synthetic Polypeptides a. Preparation of Immunogen

For preparation of a polypeptide immunogen, the synthetic polypeptides, 1:363-375, 1:417-431 and 1:237-262, were prepared as described in Example 1. Each of the synthesized polypeptides listed above were separately coupled to keyhole-limpet hemocyanin (KLH) (Sigma) using glutaraldehyde. For the coupling procedure, equivalent masses of each polypeptide was separately admixed with KLH and maintained in 20 mM phosphate and 150 mM NaCl at pH 7.2 (PBS) containing 0.075% glutaraldehyde at room temperature for 16 hours. The admixtures containing coupled polypeptides were then separately dialyzed in dialysis tubing having a molecular weight cutoff of 1000 against 100 volumes of PBS with one change. Following dialysis, equal volumes of each polypeptide-KLH carrier conjugate (300 ug/150 ul) and Freund's complete adjuvant were emulsified and injected into rabbits as described below. b. Immunization and Collection of Polyclonal Antisera

The three emulsified polypeptide-KLH immunogens prepared in Example 5a were injected into three New Zealand white rabbits after pre-immune serum from each was collected. The immunogens were injected intradermally into four sites in the back and one site above each limb. After 2 weeks, the three polypeptide-KLH conjugates were emulsified in Freund's incomplete adjuvant and the injected were repeated subcutaneously. The animals were challenged every month thereafter and sera were collected weekly starting six weeks after the initial immunization. The collected blood samples were stored at 4C for 12 hours, after which time the samples were centrifuged at 3000 X g for 20 minutes. The resultant supernatants containing anti-polypeptide antibodies were collected and stored at -20C. These supernatants were used as described below to check antibody titers against the polypeptides from which the antibodies were generated and against Factor X.

Polypeptides 366-373* (SEQ ID NO 18), 1:422-430 and 238-246* (SEQ ID NO 7) are also separately prepared as immunogens by conjugation as described in Example 2a. Immunization of separate rabbits for the production of antisera against each of the polypeptides is performed as described herein. The resultant antisera are then screened as described below for immunoreactivity. c. Radioimmunoassay to Screen Antisera Immunoreactivitv

The polypeptide antibody titers and immunospecificity in sera collected from rabbits in Example 6 were determined in a solid-phase radioimmunoassay (RIA) . The antigens used in the RIA included the three polypeptides, 1:363-375, 1:417-431 and 1:237-262, and purified Factor X prepared in Example 2. For the RIA, the polypeptides and Factor X were diluted to a concentration of 2.5 ug/ml in PBS and 100 ul of the diluted antigens were separately admixed to empty wells of polystyrene microtiter plates. The plates were maintained for 16 hours at 4C to permit the antigens to become operatively affixed to the well walls. The plates were then washed three times with SPRIA buffer (PBS containing 0.5% Tween 20, 1 unit/ml aprotinin, 10 mg/ml BSA and 0.02% sodium azide. Non-specific protein-binding sites were blocked by admixing 1% (v/v) BSA in PBS into each well and maintaining for 30 minutes at room temperature. After removal of this solution, 100 ul of rabbit nonimmune or specific antisera, serially diluted in SPRIA buffer, were admixed for 90 minutes at 37C to allow formation of solid-liquid phase immunoreaction products.

The wells were then washed three times with SPRIA buffer and maintained with 100 ul of ¹²⁵I-goat anti-rabbit IgG (0.1 ug/ml) for 90 minutes at 37C. The unbound radioactivity was removed, the plates were washed with SPRIA buffer and dried, the wells were cut out and counted in a gamma counter. Rabbit antisera which exhibited significant immunoreactivity as compared to pre-immune sera toward polypeptides were further purified and analyzed as described below. Rabbit antisera which are obtained in Example 6 against polypeptides, 366-373* (SEQ ID NO 18) , 1:422-430 and 238-246* (SEQ ID NO 7), are screened for immunoreactivity to the respective polypeptides from which the antibodies are raised as described above. Rabbit antisera which exhibit significant immunoreactivity as compared to pre-immune sera toward polypeptides are further purified and analyzed as described below.

7. Purification of Anti-Polypeptide Antibodies Purification of the IgG fraction from rabbit antisera prepared and tested in Example 6 which showed significant immunoreactivity towards the three polypeptides, 1:363-375, 1:417-431 and 1:237-262, was achieved by affinity chromatography on protein

A-agarose. Each rabbit anti-serum was separately admixed with an equal volume of 100 mM Tris-HCl at pH 9.0, 2 M NaCl and 0.1% sodium azide. The resultant admixtures were applied to individual 12 ml protein A-agarose columns. Each column was washed with four column volumes of 50 mM Tris-HCl at pH 9.0 containing 1 M NaCl and 0.05% sodium azide (TBS) to remove unbound proteins from the column. The immobilized rabbit IgG on each column was then eluted with two column volumes of 100 mM glycine-HCl at pH 3.0.

Protein was monitored by absorbance at 280 nm and the IgG concentrations were determined from the extinction coefficient of 13.5. Specific antibodies from each column were purified by passing the IgG fraction over individual 25 ml columns of Factor X coupled to agarose (2 mg/ml) by the CNBr activation method as described by March et al. , Anal. Biochem.. 60:149-152 (1974) . Each column was washed as described above and immobilized IgG was eluted with 2 M NH^SCN. Eluted antibody from each column was then dialyzed extensively against TBS at pH 7.5 and concentrated by ultrafiltration using an Amicon P-10 membrane. These immunoaffinity-purified anti-polypeptide antibodies are designated anti-1:363-375, anti-1:417-431 and anti-1:237-262.

Purification of antibodies which are raised against polypeptides, 366-373* (SEQ ID NO 18) , 1:422-430 and 238-246* (SEQ ID NO 7), are affinity purified as described above to form immunoaffinity- purified antibodies. a. Specificity and Affinity Analysis of

Immunoaffinity Purified Anti-Polvpeptide

Antibodies

The specificity of immunoaffinity purified anti-polypeptide antibodies, anti-1:363-375, 1:417-431 and 1:237-262, towards the corresponding polypeptides and Factor X was evaluated by measuring the binding in solid-phase ELISA of each antibody to above immobilized polypeptides. For the ELISA assay, 50 μM of the polypeptide and 10 μg/ml of Factor X protein were admixed into separate wells of microtiter plates as described in Example 6. After blocking the polypeptide- or protein-coated wells as described in Example 5c, 50 μl of 2 μg/ml of each immunoaffinity purified anti-polypeptide antibodies in maintenance buffer were separately admixed into each well and maintained for one hour at 37C to form an immunoreaction product. The detection and measurement of specific immunoreaction products was accomplished by admixture of streptavidin-alkaline-phosphatase followed by PNPP.

The immunoaffinity-purified antibodies prepared in Example 6 exhibited specific immunoreactivity towards the polypeptide against which the antibody was raised and also towards native Factor X protein. The separate antibodies did not cross-react with any polypeptides other than the one which was used as the immunogen. Thus, the immunoaffinity-purified polyclonal antibodies raised against polypeptides 1:₃63-375, 1:417-431 and 1:237-262 recognized specific regions on the Factor X protein corresponding to the polypeptides.

The three immunoaffinity-purified antibodies were also evaluated for their ability to inhibit the binding of ¹²⁵I-Factor X to THP-1 cells. The assays were performed as described in Example 3. Increasing concentrations of the three sequence-specific immunoaffinity-purified antibodies were premaintained with 15 nM ¹²⁵I-Factor X, prepared as described in Example 2, for 30 minutes at 22C before addition to suspensions of N-fMLP-stimulated monocyte THP-1 cells, prepared as described in Example 3. Fifty per cent inhibition of ¹²⁵I-Factor X binding was achieved with 10 μg/ml of each antibody to the three inhibitory polypeptides. Under the same experimental conditions, a sequence specific antibody to the activation polypeptide of Factor X corresponding to amino acid residue positions 21-41 was ineffective. Thus, these experiments with sequence-specific immunoaffinity- purified antibodies to each of the three polypeptides which blocked the binding of Factor X to Mac-1 provide independent evidence for specificity of recognition.

The immunoaffinity-purified antibodies raised against polypeptides, 366-373* (SEQ ID NO 18), 1:422- 430 and 238-246* (SEQ ID NO 7) are screened by ELISA and inhibition of Factor X binding experiments as described above.

8. Preparation of Monoclonal Antibodies a. Anti-polvpeptide The polypeptides designated 1:363-375, 1:417-431, 1:237-262, 366-373* (SEQ ID NO 18); 1:422- 430, and 238-246* (SEQ ID NO 7) are prepared as immunogens according to Example 6. Separate Balb/c ByJ mice (Scripps Clinic and Research Foundation Vivarium, La Jolla, CA) are immunized intraperitoneally (i.p.) with 50 μg of each of the prepared polypeptide-KLH immunogens in complete Freund's adjuvant followed by a second and third immunization using the same polypeptide-KLH immunogens, each about three weeks apart, in incomplete Freund's adjuvant. The mice receive a boost of 50 μg of each of the prepared polypeptides intravenously (i.v.) in normal saline 4 days prior to fusion and a second similar perfusion boost one day later.

The animals so treated are sacrificed and the spleen of each mouse is harvested. A spleen cell suspension is then prepared. Spleen cells are then extracted from the spleen cell suspension by centrifugation for about 10 minutes at 1000 r.p.m. , at 23C. Following removal of the resultant supernatant, the cell pellet is resuspended in 5 ml cold ammonium chloride (NH₄C1) lysing buffer, and is maintained for about 10 minutes.

Ten ml of Dulbecco's Modified Eagle Medium (DMEM) (GIBCO) and HEPES [4-(2-hydroxyethyl)-1- piperidineethanesulfonic acid] buffer are admixed to the lysed cell suspension to form an admixture, and that admixture is centrifuged for about 10 minutes at 1000 r.p.m. at 23C.

After the resultant supernatant is decanted, the pellet is resuspended in 15 ml of DMEM and HEPES and is centrifuged for about 10 minutes at 1000 r.p.m. at 23C. The above procedure is repeated. The pellet is then resuspended in 5 ml DMEM and HEPES. An aliquot of the spleen cell suspension is then removed for counting. Fusions are accomplished in the following manner using the non-secreting mouse myeloma cell line P3X63Ag 8.653.1, a subclone of line P3x63Ag 8.653 (ATCC 1580). With a myeloma to spleen cell ratio of about 1 to 10 or about 1 to 5, a sufficient quantity of myeloma cells are centrifuged into a pellet, washed twice in 15 ml DMEM and HEPES, and then centrifuged for 10 minutes at 1000 r.p.m. at 23C.

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

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

(hypoxanthine/aminopterin/thymidine) medium are admixed to the flasks.

Three days after fusion, viable cells are plated out in 96-well tissue culture plates at about 2 x 10⁴ 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 are fed seven days after fusion with HAT medium and at approximately 4-5 day intervals thereafter as needed with HT medium. Growth is followed microscopically and culture supernatants are collected about two weeks later. The culture supernatants are subsequently assayed for the presence of peptide-(390-404) specific antibody by solid-phase ELISA as described in Example 6c or by solid-phase radioimmunoassay (RIA) described below.

For screening by RIA, 50 μl of PBS containing 5 μg/ml of each of the prepared polypeptide-KLH immunogens are admixed into the wells of microtiter plates. The plates are maintained overnight (about 16 hours) at 4C to permit the separate polypeptide-KLH immunogens to adhere to well walls. After washing the wells four times with SPRIA buffer, 200 μl of SPRIA buffer containing 3% normal goat serum and 3% BSA are admixed to each well to block excess protein binding sites. The plates are maintained for 30 minutes at 20C, the wells emptied by shaking, and subsequently blotted dry to form a solid-support, i.e., a solid matrix to which each of the polypeptide-KLH immunogens are 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 37C to permit formation of solid-phase immunoreaction products. After washing the wells as previously described, 50 μl of ¹²⁵l- labeled goat anti-mouse IgG at 0.25 μg protein per ml are admixed to each well to form a labeling reaction admixture. That admixture is maintained for 1 hour at 37C to permit formation of ¹²⁵I-labeled solid-phase immunoreaction products. After washing the wells as previously described, the amount of ¹²⁵I-labeled product bound to each well is determined by gamma detection. Hybridomas are selected from hybridoma cultures that secrete anti-polypeptide antibodies to the respective polypeptides listed above into their culture media, and further characterized as described herein. b. Purification of Monoclonal Antibody

Ascites fluids are obtained from separate sets of 10-week old Balb/c mice, which are primed with 0.3 ml of mineral oil and injected intraperitoneally with 5 x 10⁶ hybridoma cells prepared above. The average time for development of ascites is 9 days.

Following clarification by centrifugation at 15,000 x g for 15 minutes at 23C, ascites fluids produced by hybridomas are pooled and stored frozen at -20C.

Purified monoclonal antibodies directed against each of the polypeptide immunogens from the hybridomas are prepared by fast protein liquid chromatography (FPLC) using a Pharmacia Mono Q HR5/5 anion exchange column (Pharmacia) using a 0-0.5 M NaCl gradient in 10 mM Tris-HCl at pH 8.0 following directions supplied with the column. Purified Mabs were concentrated using an Amicon stirred ultrafiltration cell (Danvers, MA; PM 30 membrane) to a concentration of 1 mg/ml, dialyzed into PBS (phosphate-buffered saline at pH 7.2) and stored at -70C.

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

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Altieri, Dario C

Edgington, Thomas S Fair, Daryl S

(ii) TITLE OF INVENTION: Factor X-Derived Polypeptides and

Anti-Peptide Antibodies, Systems and Therapeutic Methods for Inhibiting Inflammation

(iii) NUMBER OF SEQUENCES: 19

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Office of Patent Counsel, The Scripps

Research Institute

(B) STREET: 10666 North Torrey Pines Road

(C) CITY: La Jolla

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 92037

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US92

(B) FILING DATE: 20-NOV-1992

(C) CLASSIFICATION:

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/798,221

(B) FILING DATE: 22-NOV-1991

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Fitting, Thomas

(B) REGISTRATION NUMBER: SCR1221P

(C) REFERENCE/DOCKET NUMBER: 34,163

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 619-554-2937

(B) TELEFAX: 619-554-6312

(2) INFORMATION FOR SEQ ID N0:1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 448 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1..139

(D) OTHER INFORMATION: /note= "Factor X Light Chain"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 140..142

(D) OTHER INFORMATION: /note- "Factor X Connecting Tripeptide"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 143..448

(D) OTHER INFORMATION: /note= "Factor X Heavy Chain"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Ala Asn Ser Phe Leu Glu Glu Met Lys Lys Gly His Leu Glu Arg Glu 1 5 10 15

Cys Met Glu Glu Thr Cys Ser Tyr Glu Glu Ala Arg Glu Val Phe Glu 20 25 30

Asp Ser Asp Lys Thr Asn Glu Phe Trp Asn Lys Tyr Lys Asp Gly Asp 35 40 45

Gin Cys Glu Thr Ser Pro Cys Gin Asn Gin Gly Lys Cys Lys Asp Gly 50 55 60

Leu Gly Glu Tyr Thr Cys Thr Cys Leu Glu Gly Phe Glu Gly Lys Asn 65 70 75 80

Cys Glu Leu Phe Thr Arg Lys Leu Cys Ser Leu Asp Asn Gly Asp Cys 85 90 95

Asp Gin Phe Cys His Glu Glu Gin Asn Ser Val Val Cys Ser Cys Ala 100 105 110 Arg Gly Tyr Thr Leu Ala Asp Asn Gly Lys Ala Cys He Pro Thr Gly 115 120 125

Pro Tyr Pro Cys Gly Lys Gin Thr Leu Glu Arg Arg Lys Arg Ser Val 130 135 140

Ala Gin Ala Thr Ser Ser Ser Gly Glu Ala Pro Asp Ser He Thr Trp 145 150 155 160

Lys Pro Tyr Asp Ala Ala Asp Leu Asp Pro Thr Glu Asn Pro Phe Asp 165 170 175

Leu Leu Asp Phe Asn Gin Thr Gin Pro Glu Arg Gly Asp Asn Asn Leu 180 185 190

Thr Arg He Val Gly Gly Gin Glu Cys Lys Asp Gly Glu Cys Pro Trp 195 200 205

Gin Ala Leu Leu He Asn Glu Glu Asn Glu Gly Phe Cys Gly Gly Thr 210 215 220

He Leu Ser Glu Phe Tyr He Leu Thr Ala Ala His Cys Leu Tyr Gin 225 230 235 240

Ala Lys Arg Phe Lys Val Arg Val Gly Asp Arg Asn Thr Glu Gin Glu 245 250 255

Glu Gly Gly Glu Ala Val His Glu Val Glu Val Val He Lys His Asn 260 265 270

Arg Phe Thr Lys Glu Thr Tyr Asp Phe Asp He Ala Val Leu Arg Leu 275 280 285

Lys Thr Pro He Thr Phe Arg Met Asn Val Ala Pro Ala Cys Leu Pro 290 295 300

Glu Arg Asp Trp Ala Glu Ser Thr Leu Met Thr Gin Lys Thr Gly He 305 310 315 320

Val Ser Gly Phe Gly Arg Thr His Glu Lys Gly Arg Gin Ser Thr Arg 325 330 335

Leu Lys Met Leu Glu Val Pro Tyr Val Asp Arg Asn Ser Cys Lys Leu 340 345 350

Ser Ser Ser Phe He He Thr Gin Asn Met Phe Cys Ala Gly Tyr Asp 355 360 365

Thr Lys Gin Glu Asp Ala Cys Gin Gly Asp Ser Gly Gly Pro His Val 370 375 380 Thr Arg Phe Lys Asp Thr Tyr Phe Val Thr Gly He Val Ser Trp Gly 385 390 395 400

Glu Gly Cys Ala Arg Lys Gly Lys Tyr Gly He Tyr Thr Lys Val Thr 405 410 415

Ala Phe Leu Lys Trp He Asp Arg Ser Met Lys Thr Arg Gly Leu Pro 420 425 430

Lys Ala Lys Ser His Ala Pro Glu Val He Thr Ser Ser Pro Leu Lys 435 440 445

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Added amino-terminal glycine residue"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "Added carboxy-terminal glycine residue"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Gly Gin Asn Gin Gly Lys Gly 1 5

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Added amino-terminal glycine residue"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "Added carboxy-terminal glycine residue"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Gly Lys Asp Gly Leu Gly Glu Tyr Thr Gly 1 5 10

(2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(Iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: Internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Added amino-terminal glycine residue"

(ix) FEATURE: (A) NAME/KEY: Region

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "Added carboxy-terminal glycine residue"

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

Gly Leu Glu Gly Phe Glu Gly Lys Asn Gly 1 5 10

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Added amino-terminal glycine residue"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "Added carboxy-terminal glycine residue"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Gly Glu Leu Phe Thr Arg Lys Leu Gly 1 5

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (II) MOLECULE TYPE: peptide (Hi) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE : internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "Added carboxy-terminal glycine residue"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Leu Leu He Asn Glu Glu Asn Glu Gly Gly 1 5 10

(2) INFORMATION FOR SEQ ID NO:7:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Added amino-terminal glycine residue"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "Added carboxy-terminal glycine residue'¹

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Gly Leu Tyr Gin Ala Lys Arg Phe Lys Val Gly 1 5 10 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "Asparagine residue substitued for valine"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 17

(D) OTHER INFORMATION: /note- "Threonine residue substituted for glutamic acid"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 18

(D) OTHER INFORMATION: /note- "Glutamic acid residue substituted for glutamine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Leu Tyr Gin Ala Lys Arg Phe Lys Val Arg Asn Gly Asp Arg Asn Thr 1 5 10 15

Thr Glu

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 7..8

(D) OTHER INFORMATION: /note- "Three amino acid residue deletion from native Factor X sequence between position 7 and 8 at Factor X 245-247"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "Glutamic acid residue substituted for vallne"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Leu Tyr Gin Ala Lys Arg Phe Glu Gly Asp Arg Asn Thr Glu Gin Glu 1 5 10 15

Glu Gly Gly

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "Asparagine residue substituted for vallne" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Leu Tyr Gin Ala Lys Arg Phe Lys Val Arg Asn Gly Asp Arg Asn Thr 1 5 10 15

Glu Gin Glu Glu Gly Gly Glu Ala Val 20 25

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 7..8

(D) OTHER INFORMATION: /note= "Three amino acid residue deletion from native Factor X sequence between positions 7 and 8 at Factor X 245-247"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "Glutamic acid residue substituted for valine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Leu Tyr Gin Ala Lys Arg Phe Glu Gly Asp Arg Asn Thr Glu Gin Glu 1 5 10 15

Glu Gly Gly Glu 20

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 4..5

(D) OTHER INFORMATION: /note= "One amino acid residue deletion from native Factor X sequence between positions 4 and 5 at Factor X 257"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "Tryptophan residue substituted for two vallne residues in native Factor X sequence at 267 and 268"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Thr Glu Gin Glu Gly Gly Glu Ala Val His Glu Val Glu Trp He 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:13:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(Iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 4..5

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "Tryptophan residue substituted for two valine residues in native Factor X sequence at 267 and 268"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Thr Glu Gin Glu Gly Gly Glu Ala Val His Glu Val Glu Trp He Lys 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Tryptophan residue substituted for two valines in native Factor X sequence at 267 and 268"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Trp He Lys His Asn Arg Phe Thr Lys Glu Thr Tyr Asp Phe Asp He 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Tyrosine residue substituted for a proline"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Tyr Glu Arg Asp Trp Ala Glu Ser Thr Leu Met Thr Gin Lys Thr Gly 1 5 10 15

He

(2) INFORMATION FOR SEQ ID NO:16:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(Iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: Internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Tyrosine residue substituted for phenyalanine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Tyx Gly Arg Thr His Glu Lys Gly Arg Gin Ser Thr Arg Leu Lys 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "Lysine residue substituted for valine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Arg Gin Ser Thr Arg Leu Lys Met Leu Glu Val Pro Tyr Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "Glycine residue substituted for alanine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Gly Tyr Asp Thr Lys Gin Glu Asp Gly 1 5

(2) INFORMATION FOR SEQ ID NO:19:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(Hi) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: Internal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Glycine residue substituted for isoleucine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Gly Asp Arg Ser Met Lys Thr Arg Gly 1 5

Claims

What Is Claimed Is:

1. A polypeptide comprising no more than 12 amino acid residues and including an amino acid residue sequence represented by the formula shown in SEQ ID NO (1:366-373), wherein said polypeptide inhibits Factor X binding to Mac-1.

2. The polypeptide of claim 1 wherein said polypeptide has an amino acid residue sequence that corresponds to the sequence shown in SEQ ID NO 1.

3. The polypeptide of claim 1 wherein said polypeptide has an amino acid residue sequence represented by a formula shown in a SEQ ID NO selected from the group consisting of (18:1-9) and (1:366-373) .

4. A polypeptide comprising no more than 14 amino acid residues and including an amino acid residue sequence represented by the formula shown in SEQ ID NO (1:423-430), wherein said polypeptide inhibits Factor X binding to Mac-1.

5. The polypeptide of claim 4 wherein said polypeptide has an amino acid residue sequence that corresponds to the sequence shown in SEQ ID NO 1.

6. The polypeptide of claim 4 wherein said polypeptide has an amino acid residue sequence represented by a formula shown in a SEQ ID NO selected from the group consisting of (19:1-9), (1:422-430) and (1:423-430) .

7. A polypeptide comprising no more than 25 amino acid residues and including an amino acid residue sequence represented by the formula shown in SEQ ID NO (1:238-246), wherein said polypeptide inhibits Factor X binding to Mac-l.

8. The polypeptide of claim 7 wherein said polypeptide has an amino acid residue sequence that corresponds to the sequence shown in SEQ ID NO 1.

9. The polypeptide of claim 7 wherein said polypeptide has an amino acid residue sequence represented by a formula shown in a SEQ ID NO selected from the group consisting of (7:1-10), (7:2-11), (7:1- 11), (8:1-18) and (1:238-246).

10. A composition comprising at least two different polypeptides, each different polypeptide having an amino acid residue sequence that is from 8 to 50 residues in length and including a sequence represented by a formula shown in a SEQ ID NO selected from different formulas in the group of formulas consisting of:

(a) (1:366-373),

(b) (1:423-430) , and (c) (1:238-246), said composition being capable of inhibiting the binding of Factor X to Mac-1.

11. The composition of claim 10 wherein said polypeptide of group (a) has an amino acid residue sequence represented by the formula shown in a SEQ ID NO selected from the group consisting of (1:363-375), (18:1-9) and (1:366-373).

12. The composition of claim 10 wherein said polypeptide of group (b) has an amino acid residue sequence represented by the formula shown in a SEQ ID NO selected from the group consisting of (1:417-431), (19:1-9), (1:422-430) and (1:423-430).

13. The composition of claim 10 wherein said polypeptide of group (c) has an amino acid residue sequence represented by the formula shown in a SEQ ID NO selected from the group consisting of (1:237-262), (7:1-10), (7:2-11), (7:1-11), (8:1-18) and (1:238- 246) .

14. The composition of claim 10 wherein said composition is a therapeutic composition and said polypeptides are dispersed in a pharmaceutically acceptable excipient in a therapeutically effective amount.

15. The composition of claim 14 wherein said therapeutically effective amount is at least 0.1 weight percent polypeptide per weight of total composition.

16. A method of inhibiting Factor Xa-mediated monocyte procoagulant activity in a patient comprising administering to said patient a physiologically tolerable composition comprising a therapeutically effective amount of a Factor X polypeptide, wherein said polypeptide has an amino acid residue sequence that is from 8 to 50 residues in length and includes a sequence represented by a formula shown in a SEQ ID NO selected from the group consisting of (1:366-373), (1:423-430) and (1:238-246) .

17. The method of claim 16 wherein said polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of (1:363-375), (18:1-9), (1:366-373), (1:417-431) , (19:1-9) , (1:422-430), (1:423-430) , (1:237-262) , (7:1-10), (7:2-11), (7:1-11) , (8:1-18), (1:238-246) and (1:238-255) .

18. The method of claim 16 wherein said composition is administered intravenously.

19. The method of claim 18 wherein said intravenous administration is via bolus injection.

20. The method of claim 16 wherein said effective amount of polypeptide is an amount sufficient to produce an intravascular concentration of from 0.05 to 1000 micromolar polypeptide.

21. A method of inhibiting Factor Xa-mediated monocyte procoagulant activity in a patient comprising administering to said patient a physiologically -In¬ tolerable composition comprising a therapeutically effective amount of an antibody, wherein said antibody comprises antibody molecules that inhibit Factor X binding to Mac-1 and that immunoreact with (a) Factor X, and with (b) a polypeptide having an amino acid residue sequence that includes a sequence represented by the formula shown in a SEQ ID NO selected from the group consisting of (1:366-373), (1:423-430) and (1:238-246); but do not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula shown in the SEQ ID NO formula (1:174- 201) .

22. The method of claim 21 wherein said composition is administered intravenously.

23. The method of claim 22 wherein said intravenous administration is via bolus injection.

24. The method of claim 21 wherein said effective amount of antibody is an amount sufficient to produce an intravascular concentration of from 0.1 to 100 micrograms antibody per milliliter of blood.

25. A method for inhibiting the interaction of leukocytes with endothelial cells thereby regulating leukocyte/endothelial cell-mediated responses in a patient comprising administering to said patient a physically tolerable composition comprising a therapeutically effective amount of a Factor X polypeptide, wherein said polypeptide has an amino acid residue sequence that is from 8 to 50 residues in length and includes a sequence represented by a formula shown in a SEQ ID NO selected from the group consisting of (1:366-373), (1:423-430) and (1:238-246) .

26. The method of claim 25 wherein said polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of (1:363-375), (18:1-9), (1:366-373), (1:417-431), (19:1-9), (1:422-430), (1:423-430), (1:237-262), (7:1-10), (7:2-11), (7:1-11), (8:1-18), (1:238-246) and (1:238-255).

27. The method of claim 25 wherein said composition is administered intravenously.

28. The method of claim 25 wherein said effective amount of polypeptide is an amount sufficient to produce an intravascular concentration of from 0.05 to 1000 micromolar polypeptide.

29. The method of claim 25 wherein said leukocyte/endothelial cell-mediated responses are responses selected from the group consisting of leukocyte recruitment, leukocyte adhesion and extravasation, hematopoiesis, antigen presentation, angiogenesis, syncytial formation and hemostasis.