WO1996024063A1 - Methods for identifying integrin antagonists - Google Patents

Abstract

The invention features methods for identifying compounds capable of inhibiting the binding of a selected integrin to a selected ligand which naturally binds the selected integrin.

Description

METHODS FOR IDENTIFYING INTEGRIN ANTAGONISTS

Background of the Invention This invention relates to methods for identifying molecules capable of interfering with certain cellular immune/inflammatory responses, particularly phagocyte-mediated tissue injury and inflammation.

Circulating phagocytic white blood cells are an important component of the cellular acute inflammatory response. It is believed that a number of important biological functions such as chemotaxis, immune adherence (homotypic cell adhesion or aggregation) , adhesion to endothelium, phagocytosis, antibody-dependent cellular cytotoxicity, superoxide, and lysosomal enzyme release are mediated by a family of leukocyte surface glycoprotein adhesion receptors known as β₂ integrins or the CD11/CD18 complex. Arnaout et al., Blood 75:1037 (1990) .

The CD11/CD18 family consists of four heterodimeric surface glycoproteins, each with a distinct a subunit (CDlla, CDllb, CDllc, or CDlld) non-covalently associated with a common β subunit (CD18) . The divalent cations Ca⁺² and Mg²⁺ are essential in the stabilization and function of the β complex. The CD11/CD18 integrins mediate the stable adhesion of leukocytes to endothelium and the subsequent transendothelial migration into inflamed organs (Hynes, Cell 69:11, 1992) . CDllb/CDlδ also mediates aggregation of phagocytes (Arnaout et al., N . Engl . J . Med .312:457, 1985) , ingestion of opsonized particles, and the generation of oxygen free radicals and release of hydrolytic enzymes in response to particulate stimuli (Arnaout et al., J . Clin . Invest . 72:171, 1983) . Inherited deficiency of CD11/CD18 integrins (Leu-CAM deficiency, LAD) results in life-threatening pyogenic infections and poor wound healing due to the inability of circulating phagocytes to extravasate into infected tissues and to clear pathogens through phagocytosis and cell-mediated killing (Arnaout, Immunol . Rev. 114:145, 1990) .

While essential for host survival, CD11/CD18 integrin-mediated influx and inflammatory functions in phagocytes often exacerbate the local pathologic lesions and tissue injury in many noninfectious disease states including hemorrhagic shock, burns, atherosclerosis and hyperacute rejection (Albeda et al., FASEB J . 8:504, 1994) . In several animal models of inflammation, monoclonal antibodies to CDllb/CD18 and other CD11/CD18 integrins markedly reduce the influx and inflammatory functions of leukocytes, thus preserving tissue integrity and host survival.

The functions of CDllb/CD18 in leukocyte extravasation and inflammation are mediated through its binding to several physiologic ligands, including iC3b, the major complement C3 opsonin (Wright et al., Proc . Nat 'l Acad . Sci . 80:5699, 1983), CD54 (intercellular adhesion molecule-1, ICAM-l (Simmons et al., Nature 331:625, 1988) , and the coagulation factors fibrinogen and factor X (Altieri et al., J . Cell . Biol . 107:1893, 1988) .

Summary of the Invention The invention features methods for identifying antagonists of integrin function. The methods entail the use of an A-domain peptide, or ligand binding fragment thereof, derived from CDllb, CDlla, CDllc, CD18 (also known as β2 ) or any of the integrin β subunits having an A-domain (e.g., βl , β3, 04, β5 , β6 , βl, and β8 ) .

In one aspect, the invention features an in vitro method of screening candidate compounds for the ability to inhibit the binding of a selected integrin to a selected ligand which naturally binds to the selected integrin, the method includes: a) measuring the binding of an A-domain peptide derived from the selected integrin to the selected ligand in the presence of the candidate compound; b) measuring the binding of the A-domain peptide derived from the selected integrin to the selected ligand in the absence of the candidate compound; c) determining whether the binding is decreased in the presence of the candidate compound; d) identifying inhibiting compounds as those which decrease the binding.

In a preferred embodiment the selected integrin is a β2 integrin. In more preferred embodiments the β2 integrin is selected from the group comprising CDlla/CD18, CDllb/CD18, and CDllc/CD18; the β2 integrin is CDllb/CD18; the β2 integrin is CDlla/CD18; the β2 integrin is CDllc/CD18. In another preferred embodiment the method of claim 2 wherein the A-domain peptide is derived from the α subunit of the selected integrin; the A-domain peptide is a CDllb A-domain peptide; the A-domain peptide is a CDlla A-domain peptide; the A-domain peptide is a CDllc A-domain peptide; the A-domain peptide is derived from the β subunit of the selected integrin; the ligand is detectably labelled.

In another aspect the invention features an in vitro method of screening candidate compounds for the ability to bind to a selected integrin, the method includes: a) measuring the binding of an A-domain peptide derived from the selected integrin to the candidate compound; - 4 - d) identifying compounds capable of binding the selected integrin as those which bind to the A-domain peptide.

In one aspect of the invention candidate antagonists (e.g., peptides, antibodies, or small molecules) are tested for their ability to bind a selected A-domain peptide (or ligand-binding portion thereof) . For example, a CDllb A-domain peptide can be immobilized on a solid support and then incubated with a detectably labelled candidate antagonist. Candidate antagonists which bind to the CDllb A-domain peptide can then be further characterized by examining whether they are capable of inhibiting the interaction between the selected A domain peptide and a ligand which naturally binds to the integrin which includes the selected A domain peptide. Thus, a candidate antagonist of CDllb/CD18 function identified by its ability to bind to CDllb A domain peptide can be examined to determine whether it is capable of inhibiting the binding of EAiC3b (a natural ligand of CDllb/CD18) and CDllb/CD18 (e.g., recombinant CDllb/CD18 expressed in COS cells) .

In another aspect the of the invention candidate antagonists (e.g., peptides, antibodies, or small molecules) are tested for their ability to inhibit the binding of a selected A-domain peptide (or ligand-binding portion thereof) to a ligand to which the integrin from which the peptide is derived naturally binds. Candidate antagonists which inhibit such a binding interaction are very likely able to inhibit the interaction between the integrin from which the A-domain was derived and the ligand. Such candidate antagonists are thus likely to be capable of interferring with an immune response mediated by interaction between the integrin and ligand. For example, a CDllb A-domain peptide can be immobilized on a solid support and then incubated with a detectably ligand (e.g., iC3b) in the presence and absence of the candidate antagonist. If binding of the CDllb A-domain peptide to iC3b is less in the presence of the candidate antagonist than in the absence of the candidate antagonist are likely capable of inhibiting the interaction between the selected A domain peptide and a ligand which naturally binds to the integrin which includes the selected A domain peptide.

In either case, the candidate ligands identified by the method of the invention can be furhter characterized using any of the in vitro and in vivo assays described herein or known to those skilled in the art.

Ligands of CDlla/CD18 include: ICAM-1, ICAM-2, ICAM-3. Ligands of CDllb/CD18 and CDllc/CD18 include: ICAM-1, ICAM-2, iC3b, fibrinogen, NIF, LPS, gp63, CD23, and other endothelial, epithelial, and neutrophil ligands. Ohter lignads of CDllb and other integrins are shown in Figure 9. In the method of the invention the ligand need not be an isolated protein. FOr example cells whic hexpress the ligand or have the ligand present on their surface can be used in the screening methods of the invention.

Molecules which antagonize one or more integrin- mediated immune responses can be useful in therapeutic interventions of inflammatory diseases.

By "ligand which naturally binds to a integrin" is meant a molecule, often a protein, whihc binds to the integrin in the course of a normally occuring cell-cell, cell-matrix, or matrix-matrix interaction.

By "derived from" an integrin is meant that the A- domain is found within that integrin.

By "A-domain peptide" is meant a sequence designated herein as an A-domain or an amino acid sequence produced by introducing one or more conservative amino acid substitutions in an amino acid sequence corresponding to the sequence corresponding to that sequence. By "naturally occuring A-domain peptide" is meant a peptide sequence designated herein as an A-domain sequence. By "ligand-binding fragment" of an A-domain peptide is meant a streach of at least 10, preferably at least 20, 30, 50, or 100 amino acids within an "A-domain peptide" which retains the ability, under standard assay condition, to bind a "ligand which naturally binds to a integrin" from which the A-domain peptide is derived.

"32 integrins" and "CD11/CD18" include all leukocyte adhesion molecules which include a CD18 subunit. By the "A domain of CDllb" is meant the amino acid sequence of CDllb from Cys¹²⁸ to Glu³²¹ or an amino acid sequence produced by introducing one or more conservative amino acid substitutions in an amino acid sequence corresponding to the sequence of CDllb from Cys¹²⁸ to Glu³²¹. "CDll/CD18-mediated immune response" includes those CDll/CD18-related functions mentioned above: chemotaxis, immune adherence (homotypic cell adhesion or aggregation) , adhesion to endothelium, phagocytosis, antibody-dependent or antibody-independent cellular cytotoxicity, and superoxide and lysosomal enzyme release. Inhibition of these immune functions can be determined by one or more of the following inhibition assays as described in greater detail below: iC3b binding, cell-cell aggregation, phagocytosis, adhesion to endothelium, and chemotaxis. As used herein, a human CDllb recombinant peptide is a chain of amino acids derived from recombinant CDllb-encoding cDNA, or the corresponding synthetic DNA.

By "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation) . By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.

Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) . Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By a "substantially pure polypeptide" is meant a polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, Rps2 polypeptide. A substantially pure CDll or CD18 polypeptide may be obtained, for example, by - 8 - extraction from a natural source (e.g., a human leukocyte) ; by expression of a recombinant nucleic acid encoding a CDll or CD18 polypeptide; or by chemical synthesis. Purity can be measured by any appropriate method, e.g., those described in column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

A polypeptide or protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.

By "substantially pure DNA" is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g. , a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. By "transformed cell" is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) polypeptide (e.g., a CDllb or CD18 polypeptide) . By "peptide homologous to an A-domain peptide" is meant any peptide of 15 or more contiguous amino acids exhibiting at least 30%, preferably 50%, and most preferably 70% amino acid sequence identity to the A- domain of CDllb.

By "detectably-labelled" is meant any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably- labelling a molecule are well known in the art and include, without limitation, radioactive labelling (e.g. , with an isotope such as ³²P or ³⁵S) and nonradioactive labelling (e.g., chemiluminescent labelling, e.g., fluorescein labelling) . By "purified antibody" is meant antibody which is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., an CDllb A domain- specific antibody. A purified CDllb A domain antibody may be obtained, for example, by affinity chromatography using recombinantly-produced CDllb A domain polypeptide and standard techniques. By "specifically binds" is meant an antibody which recognizes and binds an rps protein but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes rps protein. The peptides and heterodimeric proteins of the invention are capable of antagonizing CD11/CD18 (32 integrin) mediated immune response. CD11/CD18 mediated immune responses which it may be desirable to block include acute inflammatory functions mediated by neutrophils. The molecules of the invention are useful for treatment of ischemia reperfusion injury (e.g. , in the heart, brain, skin, liver or gastrointestinal tract) , burns, frostbite, acute arthritis, asthema, and adult respiratory distress syndrome. Peptides and heterodimeric proteins of the invention may also be useful for blocking intra-islet infiltration of macrophages associated with insulin-dependent diabetes mellitus.

The invention features a purified peptide which includes at least one extracellular region of a ,52 integrin subunit capable of inhibiting a CD11/CD18 mediated immune response, the peptide lacks the transmembrane and cytoplasmic portions of the ,92 integrin subunit. In a preferred embodiment the ,52 integrin subunit is a human ,52 integrin subunit; more preferably the ,52 integrin subunit is CDlla, CDllb, CDllc or CD18; most preferably the ,52 integrin subunit is CDllb. Preferably, the peptide includes all or part of the A domain of CDllb. More preferably the peptide includes one of the following sequences: DIAFLIDGS (SEQ ID NO: 32); FRRMKEFVS (SEQ ID NO: 33); FKILWITDGE (SEQ ID NO: 34) ; VIRYVIGVGDA (SEQ ID NO: 35); DGEKFGDPLG (SEQ ID NO: 36) ; YEDVIPEADR (SEQ ID NO: 37); DGEKFGDPLGYEDVIPEADR (SEQ ID NO: 17); NAFKILWITDGEKFGDPLGYEDVIPEADREGV (SEQ ID NO: 50) ; DGEKF (SEQ ID NO: 51). In preferred embodiments, the peptide includes the amino acid sequence YYEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO: 38) ; the peptide includes the amino acid sequence KSTRDRLR (SEQ ID NO: 15) . Preferably, the peptide includes one of the following amino acid sequences:

AYFGASLCSVDVDSNGSTDLVLIGAP (SEQ ID NO: 1) ; GRFGAALTVLGDVNGDKLTDVAIGAP (SEQ ID NO: 2) ; QYFGQSLSGGQDLTMDGLVDLTVGAQ (SEQ ID NO: 3) ; YEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO: 4) ; DIAFLIDGSGSIIPHDFRRMK (SEQ ID NO: 5) ; RRMKEFVSTVMEQLKKSKTLF (SEQ ID NO: 6);

SLMQYSEEFRIHFTFKEFQNN (SEQ ID NO: 7);

PNPRSLVKPITQLLGRTHTATGIRK (SEQ ID NO: 8);

RKWRELFNITNGARKNAFK (SEQ ID NO: 9) ; FKILWITDGEKFGDPLGYEDVIPEADR (SEQ ID NO: 10);

REGVIRYVIGVGDAFRSEKSR (SEQ ID NO: 11) ;

QELNTIASKPPRDHVFQVNNFE (SEQ ID NO: 12);

ALKTIQNQLREKIFAIEGT (SEQ ID NO: 13) ; QTGSSSSFEHEMSQE (SEQ

ID NO: 14); FRSEKSRQELNTIASKPPRDHV (SEQ ID NO: 16); KEFQNNPNPRSL (SEQ ID NO: 18) ; GTQTGSSSSFEHEMSQEG (SEQ ID

NO: 19) ; SNLRQQPQKFPEALRGCPQEDSD (SEQ ID NO: 20) ;

RQNTGMWESNANVKGT (SEQ ID NO: 21) ; TSGSGISPSHSQRIA (SEQ ID

NO: 22) ; NQRGSLYQCDYSTGSCEPIR (SEQ ID NO: 23) ; PRGRARWQC

(SEQ ID NO: 24) ; KLSPRLQYFGQSLSGGQDLT (SEQ ID NO: 25); QKSTRDRLREGQ (SEQ ID NO: 26) ; SGRPHSRAVFNETKNSTRRQTQ (SEQ

ID NO: 27); CETLKLQLPNCIEDPV (SEQ ID NO: 28);

FEKNCGNDNICQDDL (SEQ ID NO: 29); VRNDGEDSYRTQ (SEQ ID NO:

30) ; SYRKVSTLQNQRSQRS (SEQ ID NO: 31) .

Preferably, the peptide includes one or more metal binding domains of CDllb. More preferably, the metal binding domains encompass amino acids 358-412, 426-483,

487-553, and 554-614 of CDllb. Most preferably, the peptide includes one of the following sequences:

DVDSNGSTD (SEQ ID NO: 46) ; DVNGDKLTD (SEQ ID NO: 47) ; DLTMDGLVD (SEQ ID NO: 48) ; DSDMNDAYL (SEQ ID NO: 49) .

In a preferred embodiment, the peptides are soluble under physiological conditions.

In another aspect, the invention features a method of controlling phagocyte-mediated tissue damage to a human patient. The method includes administering a therapeutic composition to a patient; the therapeutic composition includes a physiologically acceptable carrier and a peptide or a heterodimer of the invention. More preferably, the method is used to control phagocyte- mediated tissue damage due to ischemia-reperfussion. - 12 -

Most preferably, the method is used to control phagocyte- mediated tissue damage to the heart muscle associated with reduced perfusion of heart tissue during acute cardiac insufficiency. In another aspect, the invention features a monoclonal antibody which is raised to a peptide or a heterodimer of the invention and which is capable of inhibiting a CD11/CD18 mediated immune response.

In another aspect, the features a human CDllb recombinant peptide.

"CD11¹⁰⁸⁹/CD¹⁸⁶⁹⁹" is a heterodimer which comprises amino acids 1-1089 of human CDll and amino acids 1-699 of CD18.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments The drawings will first briefly be described. Drawings

Figure 1 is the cDNA sequence and deduced amino acid sequence of the open reading frame of human CDllb from Arnaout et al., J . Cell . Biol . 106:2153 (1988) .

Figure 2 is a representation of the results of an immunoprecipitation assay.

Figure 3 is a representation of the results of an immunoprecipitation assay.

Figure 4 is a representation of the results of an immunoprecipitation assay.

Figure 5 is a graph of the effect of various proteins and antibodies on neutrophil adhesion to endothelium.

Figure 6 is the cDNA sequence and deduced amino acid sequence of human CDlla from Larson et al., J . Cell . Biol . 108:703 (1989) . - 13 -

Figure 7 is the cDNA sequence and deduced amino acid sequence of human CDllc from Corbi et al., EMBO J . 6:4023 (1987).

Figure 8 is the cDNA sequence of human CD18 from Law et al., EMBO J . 6:915 (1987).

Figure 9 is a schematic illustration of some of the naturally occurring ligands for various integrins. The ,5 subunit are boxed. The subunits are circled. The pairing of subunits is indicated with lines drawn between the relevant α and ,5 subunits which make up the heterodimer. In each case the some of the ligands which naturally bind the heterodimer are indicated above the line, and its tissue distribution is indicated below the line in italics. (Co = collagens; LM = laminin; FN = fibronectin; VN = vitronectin; TSP = thro bospondin; FB = fibrinogen; vWf = von Willebrand factor; OP = osteopontin; FX = factor X; CHO = carbohydrates; BSP1 = bone sialoprotein 1; L = lymphocyte; M = monocyte/macrophage; PMN = granulocytes; E = eosinophils; B = basophils; NK = natural killer cells; PLT = platelets; IEL = intestinal intraepithelial lymphocytes; PBL = peripheral blood leukocytes; L- = L-selectin negative; EPI = epithelial cells; ENDO = endothelial cells; MYO = myocytes; NEU = neural tissue; MEL = melanoma; FIB = fibroblasts) .

Figure 10 is the sequence of the A-domains of ,51 (SEQ ID NO: 59); ,52 (SEQ ID NO: 58); ,53 (SEQ ID NO: 57); ,54 (SEQ ID NO: 56); ,55 (SEQ ID NO: 55) ; ,56 (SEQ ID NO: 54); ,57 (SEQ ID NO: 53) ; ,58 (SEQ ID NO: 52). In each case the sequences between "A" and "B" (each indicated by arrows) represent full length A-domain. A- domain fragments include: the sequences between "A" and "C" (both indicated by arrows) ; the sequences between "D" and "C" (both indicated by arrows) ; and the sequences between "D" and "B" (both indicated by arrows) . Figure 11 is the sequences of the A-domains of CDlla (SEQ ID NO: 61) and CDllc (SEQ ID NO: 60) .

Figure 12 is the sequences of certain CDllb fragments (SEQ ID NO: 62 TO SEQ ID NO: 78 inclusive) employed in certain binding experiments. Peptides

Each member of the ,52 integrin family is a heterodimer consisting of two subunits: a CDll subunit (with at least three variants designated CDlla, CDllb, and CDllc) and a CD18 subunit. Each subunit includes a transmembrane anchor which connects a cytoplasmic segment to an extracellular segment. The two subunits interact to form a functional heterodimer. As described in greater detail below, the extracellular segments of the ,52 integrin subunits contain various functional domains. Without wishing to bind myself to a particular theory, it appears that the peptides of the invention antagonize CDll/CD18-mediated immune responses by competitively inhibiting binding of leukocytes bearing a member of the ,5₂ integrin family to the respective binding partners of that family. Specifically, the peptides of the invention include an immune-response inhibiting extracellular segment of any one of the ,52 integrin subunits —CDlla, CDllb, CDllc, CD18— or a heterodimer composed of a portion of an α (CDlla, CDllb, or CDllc) subunit together with a portion of a ,5 subunit (CD18) . Candidate ,52 integrin subunits can be evaluated for their ability to antagonize CDll/CD18-mediated immune responses by any of several techniques. For example, subunits may be tested for their ability to interfere with neutrophil adhesion to endothelial cells using an assay described in detail below. Specific regions of the ,52 integrin subunits can be evaluated in a similar manner. Any extracellular region of a ,52 integrin subunit may be screened for its ability to interfere with - 15 -

CD11/CD18 mediated immune response. Regions of CDll whose sequences are conserved between two or more subunits are preferred candidates for antagonizing CD11/CD18 - mediated immune response. For example, the A domain (corresponding to Cys¹²⁸ to Glu³²¹ of CDllb) is conserved between CDlla, CDllb, and CDllc. The A domain is 64% identical in CDllb and CDllc and 36% homologous between these two subunits and CDlla. This domain is also homologous to a conserved domain in other proteins involved in adhesive interactions including von

Willebrand's factor, cartilage matrix protein, VLA2, and the complement C3b/C4b - binding proteins C2 and factor B. The extracellular portions of CDlla, CDllb and CDllc include seven homologous tandem repeats of approximately 60 amino acids. These repeats are also conserved in the α subunits of other integrin subfamilies (e.g., fibronectin receptor) . Arnaout et al., Blood 75:1037 (1990) .

Regions of CD18 which are conserved among ,5 integrin subunits (i.e., the ,5 subunits of ,51, ,52 and ,53 integrins) are also good candidates for regions capable of interfering with CD11/CD18 - mediated immune response. For example, CD18 has four tandem repeats of an eight- cysteine motif. This cysteine-rich region is conserved among ,5 subunits. Just amino terminal to this cysteine rich region is another conserved region, 247 amino acids long, which is conserved in several integrin ,5 subunits.

Fig. 6 depicts the cDNA sequence of human CDlla (SEQ ID NO: 39) ; Fig. 7 depicts the cDNA sequence of human CDllc (SEQ ID NO: ) ; Fig. 8 depicts the cDNA sequence of CD18 (SEQ ID NO: 41).

DNA molecules encoding all or part of CDlla, CDllb, CDllc or CD18 can be obtained by means of polymerase chain reaction amplification. In this technique two short DNA primers are used to generate multiple copies of a DNA fragment of interest from cells known to harbor the mRNA of produced by the gene of interest. This technique is described in detail by Frohman et al., Proc . Nat 'l Acad Sci . USA 85:8998 (1988) . Polymerase chain reaction methods are generally described by Mullis et al. (U.S. Patent Nos. 4,683,195 and 4,683,202) .

For example, to clone a portion of CDlla, the known sequence of CDlla is used to design two DNA primers which will hybridize to opposite strands outside (or just within) the region of interest. The primers must be oriented so that when they are extended by DNA polymerase, extension proceeds into the region of interest. To generate the CDlla DNA, polyA RNA is isolated from cells expressing CDlla. A first primer and reverse transcriptase are used to generate a cDNA form the mRNA. A second primer is added; and Taq DNA polymerase is used to amplify the cDNA generated in the previous step. Alternatively, the known sequences of CDlla, CDllb, CDllc and CD18 can be used to design highly specific probes for identifying cDNA clones harboring the DNA of interest. A cDNA library suitable for isolation of CDlla, CDllb, and CDllc DNA can be generated using phorbol ester-induced HL-60 cells (ATCC Accession No. CCL 240) as described by Corbi et al. (EMBO J . 6:4023, 1987) and Arnaout et al., Proc . Nat 'l Acad Sci . USA 85:2776, 1988) ; CD18 DNA can be isolated from a library generated using U937 cells (ATCC Accession No. CRL 1593) as described by Law et al. (EMBO J . 6:915, 1987) . These cell lines are also suitable for generating cDNA by polymerase chain reaction amplification of mRNA as described above. Isolation of a Human CDllb cDNA clone.

A 378 base pair (bp) cDNA clone encoding guinea pig CDllb was used as a probe to isolate three additional cDNA clones from a human monocyte/lymphocyte cDNA library as described in Arnaout et al., Proc . Nat 'l . Acad . Sci . USA 85:2776 (1988) ; together these three clones contain the 3,048 nucleotide sequence encoding the CDllb gene shown in Fig. 1 (SEQ ID NO: 40). Arnaout et al., J. Cell . Biol . 106:2153 (1988).

In order to express CDllb, a mammalian expression vector was constructed by assembling the above-described three cDNA clones. Appropriate restriction enzyme sites within the CDllb gene can be chosen to assemble the cDNA inserts so that they are in the same translation reading frame. Arnaout et al., J. Clin . Invest . 85:977 (1990). A suitable basic expression vector can be used as a vehicle for the 3,048 bp complete cDNA fragment encoding the human CDllb peptide; the recombinant cDNA can be expressed by transection into, e.g., COS-1 cells, according to conventional techniques, e.g., the techniques generally described by Aruffo et al., Proc . Nat 'l . Acad . Sci . USA 84:8573 (1987) or expressed in E . coli using standard techniques. Smith et al., Gene 67:31 (1988) . Isolation of CDllb Peptide from Mammalian Cells

The CDllb protein can be purified from the lysate of transfected COS-1 cells, using affinity chromatography and lentil-lectin Sepharose and available anti-CDllb monoclonal antibody as described by Pierce et al. (1986) supra and Arnaout et al., Meth . Enzymol . 150:602 (1987).

If the desired CDllb peptide is shorter than the entire protein, DNA encoding the desired peptide can be expressed in the same mammalian expression vector described above using the selected DNA fragment and the appropriate restriction enzyme site, as outlined above. The selected DNA fragment may be isolated according to conventional techniques from one of the CDllb cDNA clones or may be synthesized by standard polymerase chain reaction amplification, as described above. See also Saiki et al., (Science 239:487, 1988) . Characterization of the CDllb Polypeptide The coding sequence of the complete CDllb protein is preceded by a single translation initiation methionine. The translation product of the single open reading frame begins with a 16-amino acid hydrophobic peptide representing a leader sequence, followed by the NH₂-terminal phenylalanine residue. The translation product also contained all eight tryptic peptides isolated from the purified antigen, the amino-terminal peptide, and an amino acid hydrophobic domain representing a potential transmembrane region, and a short 19-amino acid carboxy-terminal cytoplasmic domain (Fig. 1 illustrates the amino acid sequence of CDllb; SEQ ID NO: 43) . The coding region of the 155-165 kD CDllb (1,136 amino acids) is eight amino acids shorter than the 130-150 kD alpha subunit of CDllc/CD18 (1,144 amino acids) . The cytoplasmic region of CDllb contains one serine residue that could serve as a potential phosphorylation site. The cytoplasmic region is also relatively rich in acidic residues and in proline (Fig. 1) . Since CDllb/CD18 is involved in the process of phagocytosis and is also targeted to intracellular storage pools, these residues are candidates for mediating these functions. The long extracytoplasmic amino-terminal region contains three or four metal-binding domains (outlined by broken lines in Fig. 1) that are similar to Ca²⁺-binding sites found in other integrins. Each metal binding site may be composed of two noncontiguous peptide segments and may be found in the four internal tandem repeats formed by amino acid residues 358-412, 426-483, 487-553, and 554-614. The portion of the extracytoplasmic domain between Tyr⁴⁶⁵ and - 19 -

Val⁴⁹² is homologous to the fibronectin-like collagen binding domain and IL-2-receptor. The extracytoplasmic region also contains an additional unique 187-200 amino acid domain, the A .domain, between Cys¹²⁸ to Glu³²¹, which is not present in the homologous (o) subunits of fibronectin, vitrorectin, or platelet lib/Ilia receptors. This sequence is present in the highly homologous CDllc protein (α of pl50,95) with 64% of the amino acids identical and 34% representing conserved substitutions. Arnaout et al., J . Cell Biol . 106:2153, 1988; Arnaout et al. Blood 75:1037 (1990). It is known that both CDllb/CD18 and CDllc/CD18 have a binding site for complement fragment C3 and this unique region may be involved in C3 binding. This region of CDllb also has significant homology (17.1% identity and 52.9% conserved substitutions) to the collagen/heparin/platelet Gpl binding regions of the mature von Willebrand factor (domains A1-A3) . The A domain is also homologous to a region in CDlla. Larson et al., J . Cell Biol . 108:703 (1989) . The A domain is also referred to as the L domain or the I domain. Larson et al., supra (1988) ; Corbi et al., J . Biol . Chem . 263:12,403 (1988) . CDllb Peptides

The following peptides can be used to inhibit CDllb/CD18 activity: a) peptides identical to the above-described A domain of CDllb, or a portion thereof, e.g., DIAFLIDGS (SEQ ID NO:32), FRRMKEFVS (SEQ ID NO:33), FKILWITDGE (SEQ ID NO:34) , DGEKFGDPLGYEDVIPEADR (SEQ ID NO:17), or VIRYVIGVGDA SEQ ID NO:35) ; b) peptides identical to the above-described fibronectin-like collagen binding domain, or a portion thereof, e.g., YYEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO: 38); c) peptides identical to one or more of the four metal binding regions of CDllb, or a portion thereof, e.g., DVDSNGSTD (SEQ ID N0:46) , DVNGDKLTD (SEQ ID NO:47) , DLTMDGLVD (SEQ ID NO:48), DSDMNDAYL (SEQ ID NO:49); d) peptides substantially identical to the complete CDllb; or e) other CDllb domains, e.g. KSTRDRLR (SEQ ID NO:15).

Also of interest is a recombinant peptide which includes part of the A domain, e.g,

NAFKILWITDGEKFGDPLGYEDVIPEADREGV (SEQ ID NO: 50) . The A domain binds iC3b, gelatin, and fibrinogen and binding is disrupted by EDTA. The A domain also binds both Ca²⁺ and Mg²⁺. This result unexpected since the A domain lies outside of the region of CDllb previously predicted

(Arnaout et al., J. Cell Biol . 106:2153, 1988; Corbi et al., J . Biol . Chem . 25:12403, 1988) to contain metal binding sites. Protein Sequences Kishimoto et al., Cell 48:681 (1987) disclose the nucleotide sequence of human CD18. Arnaout et al., J^". Cell Biol . 106:2153 (1988); Corbi et al., J . Biol . Chem . 263:12403 (1988); and Hickstein et al., Proc . Nat 'l . Acad . Sci . USA 86:275 (1989) disclose the nucleotide sequence of human CDllb. Larson et al., J . Cell . Biol . 108:703 (1989) disclose the nucleotide sequence of CDlla. Corbi et al., EMBO J . 6:4023 (1987) disclose the nucleotide sequence of CDllc. Moyle et al., J . Biol . Chem . 269:10008, 1994 discloses the sequence of Ancylostoma caninum neutrophil adhesion inhibitor) . The sequences of the various ,5 subunits are provided by the following references: ,51 (Argraves, W.S. et al., (1989) Cell 58, 623-629); ,52 (Kishimoto, T.K. et al., (1987) Cell 48, 681-690); ,53 (Fitzgerald, L.A. et al., (1987) J. Biol . Chem . 262, 3936-3939); ,54 (Suzuki, S. et al., (1990) EMBO J . 9, 757-763); ,55 (McLean, J.W. et al., (1990) J . Biol . Chem . 265, 17126-17131); ,56 (Sheppard, D. et al., (1990) J . Biol . Chem . 265, 11502-11507) ; ,57 (Yuan Q. et al., (1990) Int . Immunol . 2, 1097-1108) ; ,58 (Moyle et al., (1991) J . Biol . Chem . 266, 19650) . Identification of Antagonists

The screening methods of the invention employ an intact integrin A-domain or a ligand-binding fragment thereof. The A-domain of CDllb is described above. The A-domains CDlla and CDllc are depicted in Figure 11. The A-domains of integrin ,5 subunits ,51, ,52, ,53, ,54, ,55, ,56, ,57, and ,58 are presented in Figure 10. These A-domains, or ligand binding fragments thereof, can be used in the methods of the invention to identify antagonists of immunological reactions mediated by their corresponding integrin. Thus, CDllb and ,52 A-domain (or ligand-binding fragments thereof) are useful for identifying antagonists of CDllb/CD18 mediated reactions. In assays requiring the use of a ligand which binds the integrin, the preferred ligand is a ligand which is a naturally- occurring ligand of the integrin. A naturally-occurring ligand of an integrin is a ligand which interacts with the integrin as part of an cell-cell, cell-matrix, or matrix-matrix interaction. Figure 9 is a schematic illustration of the subunit composition of a number of integrins. Also shown in Figure 9 are some of the ligands which naturally bind each integrin.

The experiments described are specific examples of the identification of antagonists of cell-cell, cell- matrix, or matrix-matrix interactions mediated by integrins which include an A-domain using the methods of the invention. In first series of experiments demonstrate that an antagonist of CDllb/CDlδ, Ancylostoma caninum neutrophil adhesion inhibitor (NIF) can be identified using a screening method employing the CDllb A-domain. In the second series of experiments screening methods of the invention are used to identifying a ligand-binding fragment of CDllb A-domain which antagonizes binding of complement iC3b to CDllb/CD18. These examples are meant to illustrate, not limit, the invention.

The screening methods of the invention can be used to quickly screen libraries of peptides, antibodies, or small molecules to identify antagonists.

Also described are a number of assays which can be used to further characterize antagonists identified by the methods of the invention.

Binding of NIF to the CDllb A-domain For this experiment recombinant CDllb A domain

(rCDllbA) and recombinant CDlla A domain (rllaA) were expressed as GST fusion proteins as described below, and used as such or after thrombin cleavage.

The recombinant peptides were immobilized and the binding of biotinylated recombinant NIF was measured.

Biotinylated rNIF bound directly and specifically to immobilized rllbA. Binding of rNIF to this domain was characterized by a rapid on rate, and a slow off rate, that were almost identical to those characterizing NIF binding to whole neutrophils (see below) . NIF binding to immobilized rA-domain was specific and saturable.

Scatchard analysis of this binding yielded an apparent K_d of -1 nM, similar to that obtained when the neutrophil- bound native CDllb/CD18 was used (see below) . In western blots, biotinylated NIF bound directly to rllbA but not to rllaA, and binding to rllbA was inhibited completely by the mAb 107, and partially by OKM9, but not by 44,

904 or TS1/22, indicating the specificity of rllbA-NIF interactions. The following experiments demonstrate that binding of NIF to recombinant CDllb A-domain is metal dependent.

Binding to rCDllb A-domain was measured as described below

Binding of NIF to immobilized rllbA required divalent cations, as it was blocked in the presence of EDTA. EDTA - 23 - was also able to completely reverse rllbA-NIF interaction even when added one hour after the complex is formed. NIF bound to rllbA in VBSG buffer under these conditions, and binding was not significantly affected by Chelex treatment of VBSG^"" or by addition of Ca²⁺, Mg²⁺, or Mn²⁺ each at ImM. Addition of EGTA at 1 mM to the VBSG^" buffer reduced NIF binding only marginally, indicating that binding can occur in the absence of Ca²⁺. As the other cations (e.g. Mg²⁺, Mn²⁺) cannot be selectively chelated, we cannot exclude that binding of NIF to rllbA can occur in presence of Ca²⁺ alone. Since binding is abolished by EDTA, trace amounts of other divalent cations (derived from the buffer salts, gelatin, glucose or BSA) are essential. The divalent cations appear to be required at least at the level of the A-domain, since the mutant rllbA (D140GS/AGA) that lacks the metal binding site(s) did not bind NIF even in the presence of 1 mM each of Ca²⁺ and Mg²⁺. Binding of NIF to the A- do ain was not affected by temperature as in whole cells. Fluid-phase rllbA, but not its fusion partner GST, abolished biotinylated rNIF binding to human neutrophils or to immobilized rllbA in a dose dependent manner, with half-maximal inhibition seen at ~1 nM in each case, reflecting the lack of significant structural differences between the adsorbed and soluble forms of rllbA.

To identify the site in rllbA involved in NIF binding, eleven overlapping peptides spanning the A- domain were synthesized and tested for their ability to inhibit NIF binding to immobilized rllbA. We found that the two contiguous peptides (A6 and A7) inhibited binding of rNIF to rCDllb A-domain dramatically. A scrambled form of A7 had no such effect. Two additional peptides (Al and A12) , located at the beginning and end of the domain had moderate and weak inhibitory effects respectively. Dose response curves revealed that while combining A6 and A7 each at 161 mg/ml (80-115 mM) achieved complete inhibition of biotinylated NIF binding to rllbA, addition of Al (but not A12) produced a shift in the binding curve to the left suggesting that Al within the recombinant A-domain also contribute to NIF- rllbA interaction. Some peptides (A7, A7M, A3, All) adsorbed well to microtiter plates, allowing an assessment of the direct binding of rNIF to these peptides. Biotinylated rNIF bound to immobilized A7 peptide but not to A3 and All. Binding of NIF to A7 was not affected when the aspartate residue at position 242 (involved in metal coordination in rllbA and CDllb/CD18) is replaced with alanine. Direct binding of rNIF to A6, Al, A12 could not be tested because these peptides did not absorb to plastic wells.

Generation and purification of CDll A-domain recombinant proteins: The GST-fusion proteins were produced in Escherichia coli using standard methods (see Machishita et al., Cell 72:859, 1993; Ueda et al., Proc . Nat 'l Acad . Sci USA 91:10684, 1994). The GST fusion proteins were purified by affinity chromatography using the method of Smith et al. (Gene 67:31, 1988) and used as fusion proteins or cleaved with thrombin (Gene 67:31, 1988) to release the A-domains. Recombinant purified NIF (rNIF) provided by Drs. Matthew Moyle and Howard R. Soule (Corvas International Inc., San Diego). A recombinant soluble form of human CD54 (containing all five Ig domains but lacking the intramembranous and cytoplasmic regions) was provided by Dr. Jeffrey Greve (Miles Research Center, West Haven, CT; Greve et al, Cell

56:839, 1989). Recombinant protein concentrations were determined using the protein assay kit from BioRad Laboratories (Melville, NY) and analyzed by Coomassie staining after electrophoresis on denaturing polyacrylamide gels (Laemmli, Nature (Lond) . 227:680, 1970) .

Each recombinant protein reacted with several blocking monoclonal antibodies (44, 904, OKM9 and 107 in the case of the rllbA, and TS1/22 and LI in the case of the rllaA; Ueda et al., Proc. Nat 'l Acad . Sci USA 91:10684, 1994), confirming the identity of the polypeptides.

Reagents. Synthetic Peptides. and Antibodies: Restriction and modification enzymes were purchased from New England Biolabs (Beverly, MA) , Boehringer Mannheim Biochemicals (Indianapolis, IN) or BRL (Gaithersburg, MD) . The vector pGEX-2T was obtained from Pharmacia LKB Biotechnology, Inc. (Piscataway, NJ) . Murine mAbs directed against human CDllb [44 (Arnaout et al., J. Clin. Invest. 72:171, 1983); 904 (Dana et al., J.

Immunol. 137:3529, 1986) ; OKM9 (Wright et al., Proc. Nat'l Acad. Sci. USA 80:5699, 1983)], CDlla [TS1/22 (Sanchez-Madrid et al., J. Exp. Med. 158:1785, 1983)], CD18 [TS1/18 (Sanchez-Madrid et al., J. Exp. Med. 158:1785, 1983)], and CDllc [L29 (Lanier et al., Eur. J. Immunol. 15:713, 1985)] were prepared as described in the cited references. The mAb 107 was prepared by immunizing BALB/c mice with pure recombinant CDllb A-domain. This mAb reacts with CDllb but not CDlla A-domain by ELISA, immunoprecipitates CDllb/CD18 from neutrophil extracts, and and binds to neutrophils by FACS analysis. Synthetic peptides can be obtained commercially and purified by HPLC according to standard techniques. In some cases selected peptides were subjected to amino acid analysis. Synthetic peptides described herein were soluble in water at 1 mg/ml.

Immobilization of Recombinant Proteins and Polypeptide: Purified rA-do ain preparations (1 μg/well) , soluble CD54, human fibrinogen (Sigma Chemical Co., St. Loius, MO) , gelatin (BioRad Laboratories) or BSA (Calbiochem-Behring Corp.) (each at 10 μg/well) or selected A-domain-derived peptides (10 μg) were added to Immulon-2 96-well microtiter plates (Dynatech Labs, Chantilly, VA) overnight. Quantitation of adsorbed wild- type and mutant A-domain and synthetic peptides was done using the mAb 44 in an ELISA, and the BCA kit (from Pierce Chemical Co. , Rockford, IL) , respectively. Wells were then washed with phosphate-buffered-saline (PBS) , pH 7.4 without metals, and blocked with 1% BSA for one hour, washed again in binding buffer and used immediately in the functional assays.

Biotinylation of recombinant NIF and Measurement of Binding to Immobilized Peptides: Recombinant NIF was labeled with sulfo-NHS-biotin as described by the manufacturer (Pierce Chemical Co.). To measure binding of biotinylated rNIF to immobilized rllbA, increasing concentrations of biotinylated rNIF in VBSG⁺⁺ (veronal- buffered saline, pH 7.4, containing 0.1% gelatin, 1 mM CaCl_2/ 1 m MgCl₂) in the absence or presence of 100-fold unlabeled rNIF, were added to A-domain-coated 96-well microtiter wells, and incubated at RT for 60 minutes. Wells were then washed, incubated with alkaline phosphatase-coupled avidin, washed again, developed with substrate and quantified colormetrically using a microplate reader. To evaluate the ability of anti-CDllb A-domain mAbs to block biotinylated NIF binding to immobilized rllbA, coated wells were preincubated with the mAbs (each at 100 mg/ml or 1:100 dilution of ascites) for 15 minutes at RT. Biotinylated NIF (50 ng/ml final concentration) was then added, and incubation continued for an additional hour. To assess the ability of fluid- phase rllbA or GST to block biotinylated NIF binding to immobilized rllbA, each was preincubated at 7 mg/ml with biotinylated NIF (50 ng/ml final concentration) in a total volume of 50 μl for 15 minutes at RT, followed by incubation of this mixture with the rllbA-coated wells for an additional hour. In experiments where the effects of divalent cations on biotinylated rNIF binding to immobilized rCDllb A-domain were measured, VBSG buffer (veronal-buffered saline, pH=7.4, containing 0.1% gelatin) containing 1 mM of Ca²⁺, Mg²⁺, Mn²⁺, EDTA, EGTA, EGTA plus 1 mM MgCl₂, or 1 mM MnCl₂. In these experiments, BSA-blocked A-domain containing wells were first washed with buffer containing 10 mM EDTA (to remove protein-bound cations) , then washed with the respective binding buffer. The effect of temperature was evaluated in the presence of the standard divalent cation mixture at 37°C, 22°C and at 4°C with saturating amounts of biotinylated rNIF (200 ng/ml) . The kinetics of rNIF-neutrophil or rNIF-A-domain interactions were determined as described by Lowenthal et al. (In Current Protocols in Immunology , Colgan et al., eds. Vol. 1:6.0.1-6.1.15, 1992). Neutrophils or immobilized rA-domains were each incubated with half- saturating concentrations of biotinylated rNIF (20 ng/ml and 40 ng/ml for neutrophils and immobilized rA-domain, respectively) , in the absence or presence of 100-fold molar excess of unlabeled rNIF at 4'C (with neutrophils) or at RT (with immobilized rA-domain) . The specific binding of biotinylated rNIF was determined at various times as described above, and plotted vs. time. The time required to reach equilibrium was one hour. The value for t^_/2 of association was determined graphically from the association plot. To determine dissociation rates, neutrophils or immobilized A- domains were incubated for one hour with the respective half-saturating concentrations of biotinylated rNIF mentioned above, in the absence or presence of 100 fold molar excess of unlabeled rNIF, at 4'C (for neutrophils) or at RT (for immobilized A-domain) . Afterwards, neutrophils were washed twice in VBSG⁺⁺ and incubated in 4 ml of this buffer on ice with shaking. At various time points, aliquots were removed, centrifuged and the amount of specifically bound rNIF measured. For immobilized rllbA- domain, wells were washed twice and incubated with 300 μl of VBSG^"1"1' per well at RT with shaking. At various time points, buffer was removed and specific binding was measured. The dissociation rates in each case were determined by plotting -ln(B/B ) versus time, where B and B_eq represent respectively the fraction of rNIF bound to cells (or to immobilized rllbA-domain) at time t, and at equilibrium. The value for t_{1 2} of dissociation was calculated according to the formula

(Lowenthal et al., In Current Protocols in Immunology, Colgan et al., eds. Vol. 1:6.0.1-6.1.15, 1992).

Characterization of the Effect of NIF Integrin Function

Having identified NIF as a protein which can bind to CDllb A-domain, a series of additional assays can be employed the characterize the effect of NIF on integrin function. These characterization assays, described in more detail below, can be used to assess any CDllb A- domain binding molecule identified using the method of the invention. Binding of NIF to Neutrophils: The time course of association of biotinylated NIF with neutrophils at 4° C (to avoid endocytosis) was performed as described below. These measurements revealed a rapid uptake, with maximum levels achieved within 60 minutes, and with a t_{1 2} at 15 minutes, and was completely inhibited in the presence of 100-fold molar excess of unlabeled NIF at each time point.

Upon washing and dilution of cells preincubated for one hour at 4°C with biotinylated rNIF, the cell- associated rNIF slowly dissociated with a t₂/ of -7.6 hours. Thus, the association of rNIF with neutrophils is reversible and characterized by rapid binding and very slow dissociation. The slow dissociation rate permitted the use of biotinylated rNIF under the conditions described to evaluate its interaction with whole cells and with protein fragments. Incubation of increasing concentrations of biotinylated rNIF with resting or activated neutrophils at 4°C, revealed a predominantly saturable component, with the non-saturable (non¬ specific) fraction (obtained in the presence of 100-fold molar excess of unlabeled rNIF) accounting for less than 10% of the total binding. A Scatchard plot of the binding data indicated a linear relationship in both resting and activated cells. Both cell types bound NIF with approximately similar affinities (apparent dissociation constants K_d, ranging from 0.35 to 1.3 nM) , suggesting that the 12-fold increase in NIF binding to activated vs resting cells is primarily due to an increase in the number of NIF binding sites induced by cell activation. Biotinylation of recombinant NIF and Measurement of Binding to Neutrophils

100 μg of rNIF were labeled with sulfo-NHS-Biotin as described above. rNIF binding to resting or stimulated human neutrophils (pretreated with 10^~6 M f- met-leu-phe, for 15 minutes at 37°C, then washed) was measured. Increasing amounts of biotinylated rNIF in the absence or presence of 100-fold molar excess of unlabeled rNIF were incubated on ice for one hour with lxlO⁶ neutrophils in VBSG⁺⁺ in a total volume of 50 ml. Cells were then washed and incubated with phycoerythrin-coupled avidin (Sigma Chemical Co.) under similar conditions, washed again, fixed in 1% paraformaldehyde in PBS, and analyzed using FACScan (Becton Dickinson Co. , Mountain View, CA) . Mean channel fluorescence for each sample was then expressed as a function of the amount of biotinylated rNIF used. Background binding of phycoerythrin-streptavidin alone to neutrophils was subtracted (2.8 fluorescent units). Specific binding was obtained by subtracting total binding from that seen in the presence of excess unlabeled rNIF, and the values plotted according to Scatchard (Ann. N.Y. Acad. Sci. 51:660, 1949). To determine the effect of unlabeled fluid-phase rllbA or GST on rNIF binding to neutrophils, each was preincubated at varying concentrations with biotinylated rNIF (20 ng/ml, final concentration) for 15 minutes on ice before addition of the mixture to neutrophils. The effect of mAbs on biotinylated NIF binding to neutrophils was assessed by preincubating the neutrophils with 100 μg/ml of each mAb at 4°C for 15 minutes before addition of biotinylated NIF (20 ng/ml) . The incubation then continued for one hour, followed by processing of cells for FACS analysis as described below. Effects of rNIF on Neutrophil Ligand Binding and Phagocytosis

The effects of rNIF on CDllb/CD18-mediated neutrophil binding to the physiologic ligands complement iC3b, fibrinogen, and CD54 were measured. rNIF inhibited binding of EAiC3b to recombinant human CDllb/CD18 (expressed in COS cells) in a dose-dependent manner with complete inhibition achieved at 3 mg/ml (IC₅₀ of - 5 nM) . rNIF also abolished iC3b-dependent phagocytosis of serum- opsonized oil red 0 particles by human neutrophils.

Binding of f-met-leu-phe-activated fluoresceinated neutrophils to microtiter wells coated with human fibrinogen or soluble CD54 was also inhibited significantly in the presence of NIF (5 μg/ml) . Inhibition of neutrophil binding to fibrinogen was incomplete even at high NIF concentrations (50 mg/ml) . CD54 binds to both CDlla/CD18 and CDllb/CD18. Complete inhibition of neutrophil-CD54 interactions therefore requires the simultaneous use of mAbs directed against both antigens. Although NIF did not inhibit neutrophil binding to CD54 when used alone, it abolished this binding when combined with an anti-CDlla mAb.

Preparation of complement C3-coated erythrocytes: Sheep erythrocytes were incubated with 1:240 dilution of rabbit anti-sheep erythrocyte antiserum (Diamedix Corp. , Miami, FL) for 30 min at 37°C to generate IgM-coated sheep erythrocytes (EA) . EAiC3b was prepared using C5- deficient human serum (Sigma Chemical Co., St. Louis, MO) at 1:10 dilution (60 min at 37°C) . EAiC3b cells were washed and stored in isotonic VBSG⁺⁺ to which Soybean Trypsin Inhibitor (STI; Worthington Biochemical Co., Freeton, NJ) was added at 1 mg/ml. EAiC3b (at 1.5 x 10⁸ cells/ml) were labeled with 5-(and-6)-carboxy fluorescein (Molecular Probes, Eugene, OR) at 1:100 dilution of a 10 mg/ml stock for 5 min on ice and washed before use in the binding studies. Recombinant CDllb/CD18 binding to EAiC3b: Binding of EAiC3b to recombinant, membrane-bound CDllb/CD18 expressed on COS cells was performed as described by Machishita et al.(Cell 72:857, 1993). To assess the effect of NIF on this interaction, EAiC3b binding was performed in the absence and presence of increasing amounts of NIF. After incubation, cells were washed, examined briefly by light microscopy then solubilized with 1% SDS-0.2 N NaOH. Fluorescence was quantified (excitation wavelength, 490 nm, emission wavelength, 510 nm) on each sample using a SLM 8000 fluorometer (SLM Instruments, Urbana, IL) as described by Machishita et al. (Cell 72:857, 1993) .

Neutrophil binding to fibrinogen and CD54: Human neutrophils were purified as described by Boyum et al. (Scand. J. Clin Lab. Invest. 97 (Suppl.) :77, 1968). - 32 -

Binding of neutrophils to CD54-coated or fibrinogen- coated 96-well microtiter plates was performed as follows: Neutrophils (8xl0⁶/ml) were labeled with 5-(and- 6) -carboxy fluorescein (Molecular Probes, Eugene, OR) at 1:100 dilution of a 10 mg/ml stock for 5 min on ice and washed in M199 medium containing an additional 1 mM MgCl₂, 1 mM CaCl₂ and 0.1% BSA (MB) before use. Fluoresceinated neutrophils (25 μl of 8xl0⁶/ml) were added to each well containing 25 μl of buffer alone or containing 2xl0^"6 M f-met-leu-phe. The plates were centrifuged at RT (800 rp in a Sorvall RT 6000B) for 30 s, and incubated for only five min at RT, to avoid cell spreading, a fact confirmed by visual inspection of the cells at the end of this incubation period. Wells were washed three times with 100 ml of MB each, examined by light microscopy, then solubilized with 1% SDS/0.2N NaOH and fluorescence quantified. To evaluate the effects of mAbs and NIF on binding, mAbs (each used at 1:100 dilution of ascites) or NIF (used at 5 mg/ml final concentration) were preincubated with fluoresceinated neutrophils for 15 minutes at 4°C prior to the binding reaction.

Phagocytosis Assays: Phagocytosis of serum opsonized oil red O (ORO) particles was performed essentially as described by Arnaout et al. (N . Engl . J . Med . 306:693, 1982) . To determine the effect of rNIF or the anti-CDllb mAb 44 on phagocytosis, rNIF (at 4 μg/ml) or 44 (at 10 μg/ml) were preincubated with neutrophils for 10 minutes at RT prior to addition of opsonized ORO. The reactants were prewarmed for 2 minutes at 37°C before mixing. Incubation was then commenced for 5 min at 37°C with continuous shaking in a water bath. The reaction was stopped by addition of 1 ml of ice-cold PBS containing 1 mM N-ethyl-maelamide (NEM) , followed by two washes. The cell pellet was examined visually for its red color (reflecting ingestion of the red oil droplets) , then solubilized with 0.5 ml of dioxane, and the amount of ORO in the extract quantified by measuring absorption at 525 nm and converted to milligrams of ORO ingested/10⁵ cells/minute. Specific uptake of ORO was determined by subtracting the background (uptake in the presence of 1 mM NEM) . Binding of NIF to CDllb/CD18

Western blots of heterodimeric CDllb/CD18 immunoprecipitated from unlabeled neutrophils were probed with biotinylated rNIF, and the pattern was compared with biotinylated CDllb/CD18 (generated by surface biotinylation of neutrophils) . This analysis showed that rNIF binds to the CDllb but not the CD18 subunit of the CDllb/CD18 heterodimer. rNIF did not bind to the other two b2 integrins CDlla or CDllc expressed on neutrophils.

To determine if CDllb/CD18 is the only receptor on the neutrophil surface that binds to NIF, several anti- CDllb mAbs known to inhibit CDllb/CD18 functions were evaluated for their ability to block the binding of biotinylated NIF to neutrophils. These studies demonstrated that mAb 107 inhibited NIF binding to neutrophils completely. Two other anti-CDllb mAbs, 44 and 904, and the anti-CDlla mAb (TS1/22) had no inhibitory effect.

Surface biotinylation. immunoprecipitation and Western blotting: Surface biotinylation of purified human neutrophils was performed on ice by incubating the cells (3xl0⁷/ml in PBS) with 0.1 mg/ml final concentration of Sulfo-NHS-Biotin (Pierce Chemcial Co.) for 30 min at 4°C. Afterwards, cells were washed twice in PBS, quenched for 15 min in RPMI on ice and washed once again in PBS. The NP-40- soluble fraction from unlabeled or biotin-labeled cells was used to immunoprecipitate ,52 integrins proteins with the anti-CDlla, b, c-specific mAbs (TS1/22, 44, L29, respectively) . Immunoprecipitates were electrophoresed on gradient 4-16% polyacrylamide gels in Laemmli buffer, electroblotted onto Immobilon-P membranes and blocked with BSA. Membranes containing immunoprecipitates from surface-biotinylated cells were then probed with HRP- coupled avidin (Sigma Chemical Co.) , while those with immunoprecipitates from unlabeled cells were first probed with biotinylated rNIF (at 1 mg/ml) , washed then re- probed with HRP-coupled avidin (Sigma Chemcial Co.). Membranes were developed using the ECL system from Amersham Corp. (Arlington Heights, IL) . NIF as a Disintegrin

Taken together the above-described experiments demonstrate that hookworm-derived NIF is a specific CDllb/CD18 antagonist that binds to neutrophils through the CDllb A-domain and inhibits their ability to recognize several CDllb/CD18 ligands and to mediate phagocytosis. The binding of NIF to the CDllb A-domain is selective, of high affinity and divalent cation- dependent. The NIF binding site in rllbA partially overlaps that of human iC3b, the major complement C3 opsonin.

Evidence supporting that CDllb/CD18 is the sole receptor on the neutrophil surface for NIF is based on four types of experiments. First, binding of biotinylated NIF to intact cells was completely blocked by an anti-CDllb/CD18 mAb. Second, probing western blots of detergent extracts from normal or ,52 integrin- deficient neutrophils with biotinylated NIF revealed a single specific band, that of CDllb, in normal cell lysates, that was lacking in the genetically-deficient cells. Third, of the three ,52 integrins immunoprecipitated from normal neutrophils, only the CDllb subunit reacted with biotinylated NIF in western blots. NIF bound to neutrophil CDllb/CD18 with high affinity (nM range) and inhibited the binding of neutrophils to the CDllb/CD18 ligands iC3b, fibrinogen and CD54. Fourth, soluble rllbA completely blocked the binding of biotinylated NIF to neutrophils. These findings indicate that NIF is a highly selective CDllb/CD18 antagonist.

Previous studies have identified several naturally-occurring proteins, so-called disintegrins, that bind to other integrins with high affinity and block integrin-mediated adhesion (reviewed in Philips et al., Ceil 65:359, 1991). Disintegrins isolated from leeches and snake venoms inhibit adhesion-dependent functions such as platelet aggregation when present in low nanomolar concentrations. The majority of disintegrins contain the tripeptide Arg-Gly-Asp and have so far been shown to bind to integrins lacking the A-domain (e.g., members of the ,51, ,53 and ,55 integrin families) . Disintegrins interact with their respective receptors through a disintegrin domain, a -60 amino acid motif with a characteristic cysteine-rich profile. NIF neither contains an Arg-Gly-Asp sequence, nor the disintegrin motif (Moyle et al., J. Biol. Chem. 269:10008, 1994) . The unique structure of NIF probably reflects different structural requirements for antagonists targeting the A- domain-containing integrins. It is interesting to note that the physiologic ligands of CDllb/CD18 such as iC3b, fibrinogen and CD54 do not contain or do not require an Arg-Gly-Asp sequence. NIF may similarly contain a novel motif with cellular counterparts functioning perhaps in regulating important physiologic interactions.

Identification of the active site in NIF involved in integrin binding should be very useful in this regard. The binding site of NIF in CDllb/CD18 is the A- domain. This conclusion is based on the following observations. First, NIF bound to rllbA directly, - 36 - specifically and with kinetics and affinity very similar to that in whole neutrophils. Second, binding of NIF to immobilized rllbA was blocked by the anti-CDllb A-domain mAb 107 or with excess unlabeled fluid-phase rllbA. Third, fluid-phase rllbA completely blocked the binding of biotinylated NIF to intact neutrophils. Treatment of hookworm disease

By producing a factor, NIF, that blocks CDllb/CD18-mediated functions in neutrophils, hookworms may be able to prevent neutrophil extravasation into infected regions and the destruction of the parasites through their phagocytic and killing abilities. Because rCDllbA inhibits NIF binding to leukocytes in the low nM range whereas its inhibition of iC3b binding to the same cells requires micromolar concentrations, rCDllbA may be useful as such or in a modified form for the treatment of hookworm infection, without producing generalized immunosuppression. Methods for Identifying Ligand Binding Portions of an Integrin A-domain

The experiments described below illustrate one systematic means for identifying a ligand binding fragment of an A-domain peptide. In this method a series of overlapping peptides spanning the A-domain are created. These peptides are then test for their ability to bind to a selected integrin ligand (preferably a naturally-occurring ligand, e.g., complement iC3b) . Both direct and indirect assays are illustrated below. In the direct assay binding of the A-domain peptide fragment to the selected ligand is measured and used as a gauge of the ligand binding ability of the peptide fragment. In the indirect assay the ability of the fragment to inhibit binding of full-length A-domain peptide to a ligand to the full-length A-domain peptide is measured and used as a gauge of the ligand binding ability of the peptide fragment.

Materials: To generate the CDlla A-domain, the respective cDNA was cloned by PCR using CDlla cDNA based oligonucleotides as described by Larson et al. (J. Cell . Biol . 108:703, 1989), inserted in-frame into the BamHl- Smal restricted pGEX-2T vector (Pharmacia) , and the ligated product purified and used to transform E . coli JM109. Individual bacterial clones containing the cloned cDNA fragment were identified by restriction analysis, and the recombinant protein expressed as a glutathione-S- transferase (GST) fusion protein, purified and released by thrombin (Michishita et al. Cell 72:857, 1993; Smith et al., Gene 67:31, 1988), and analyzed on denaturing 12% polyacrylamide gels. Synthetic peptides were obtained commercially, purified on HPLC, and selective ones were subjected to amino acd analysis.

Erythrocytes (E) coated with rabbit anti-E IgM (EA) or C3b (EAC3b) were prepared as described by Dana et al. (J. Immunol . 73:153, 1984). EAiC3b (erythrocytes coated with iC3b) were generated by treating EAC3b with purified human factors H and I, or alternatively prepared from EA using C5-deficient human serum (Sigma Chemical Co., St. Louis, MO). EAiC3b cells were washed and stored in isotonic veronal-buffered saline (VBS²⁺) , pH 7.4, containing 0.15 mM calcium-1 mM magnesium (MgCl₂+CaCl₂) and 1 mg/ml Soybean Trypsin Inhibitor (STI; Worthington Biochemical Co., Freehold, NJ) at 1.5 x 10⁸ cells/ml. EA, EAC3b or EAiC3b were labeled with 5-(and- 6)-carboxy fluorescein (Molecular Probes, Eugene, OR) as described by Michishita et al. (Cell 72:857, 1993).

Immobilization of recombinant proteins and peptides: Purified recombinant A-domain was added to Immulon-2 96-well microtiter plates (Dynatech) overnight. Wells were then washed once with phosphate-buffered- saline, pH 7.4 without metals, and blocked with 1% BSA at room temperature (RT) for one hour, followed by two washings with buffer A (composed of 60% GVBS:VBS²⁺ mixed in a 1:3 ratio; Arnaout et al., in Complement Receptor Type 3 at 602-615, Academic Press, FL) containing 1 mM MnCl₂ or MgCl₂+CaCl₂. All the peptides were stocked at 1 mg/ml in water and similarly adsorbed to Immulon-2 96- well plates. Binding of the anti-CDllb mAbs to the coated rA-domain was measured by ELISA and read using a plate reader (Molecular Dynamics) .

Erythrocyte binding assays: Fluoresceinated EAiC3b, EAC3b or EA were resuspended to 1.5xl0⁸/ml in buffer A, and added (30 ml) to wells containing immobilized proteins or peptides in a total volume of 100 ml. The plates were then briefly centrifuged to settle the erythrocytes, and allowed to incubate at 37°C for 15 minutes in a humidified incubator with 5% C0₂. For the inhibition studies, E were preincubated with each recombinant protein or pure peptide in the presence of 2% BSA for 5 minutes at RT and added to wells coated with immobilized protein or peptide without washing, unless otherwise indicated. At the end of the binding reactions, wells were washed, examined briefly by light microscopy then solubilized with 1% SDS-0.2 N NaOH. Fluorescence was quantified (excitatory wavelength, 490 nm, emission wavelength, 510 nm) using a SLM 8000 fluorometer (SLM Instruments, Urbana, IL) . In experiments where the effects of individual divalent cations were measured, Ca²⁺ and Mg²⁺ were replaced with metal-free buffers or with buffers containing each cation at 1 mM, unless otherwise indicated. The effect of temperature was evaluated in the presence of 1 mM MnCl₂ at 37°C and at 4°C.

Purification and adherence of human neutrophils: Neutrophils were purified as described by Boyum et al. (Scand . J . Clin . Lab . Med . 97 (suppl. ) :77, 1968), resuspended in divalent-cation-free Tris-HCl-saline buffer, pH 7.4 at 5xl0⁷/ml and kept on ice until used. Neutrophils (2 xlO⁵ cells/well) were allowed to adhere to 96-well plates in Iscov' s Modified Medium for one hour at 37° C, in a humidified incubator with 5% C0₂. The wells were then washed, and 5 ml of fluoresceinated EAiC3b or EA (at 1.5xl0⁸/ml) were added in the presence of 3% BSA, in a total volume of 50 ml , followed by 15 min incubation at 37° C with 5% C0₂. Wells were then washed and fluorescence quantified as described above.

Flow Cytometry: Fifteen ml of EAiC3b or EA (each at 1.5xl0⁸/ml) were incubated with 15 mg of biotinylated A7 or control peptides in 100 ml of buffer A containing 1 mM MnCl₂ at RT for 10 min and washed once. Streptavidin conjugated phycoerythrin (Sigma) was added to the cell suspension at 1 mg/ml and incubated for 15 min at RT. Washed E were then analyzed by a fluorescence activated cell sorter from Becton Dickinson. The CDllb A-domain contains an iC3b binding site: The ability of fluoresceinated EAiC3b to bind to a water soluble rCDllb A-domain was examined. The recombinant domain reacted with several mAbs known to inhibit the function of CR3 in whole cells (mAbs: 44, OKM9, and 904). The human rA-domain was immobilized onto 96-well microtiter plates, and incubated with fluoresceinated EAiC3b, EAC3b or EA at 37°C in the presence of divalent cations. After several washes, the number of bound erythrocytes were quantified using a fluorometer. The rA- domain bound to EAiC3b but not to EAC3b or to EA. The percentage of bound EAiC3b increased progressively as a function of the concentration of the rA-domain used to coat the microtiter wells. Optimal binding occurred upon addition of 20 mg of A-domain, and using 30 ml of EAiC3b (at 1.5xl0⁸/ml) per well. Under these conditions EAiC3b binding was easily visible by the naked eye, and was - 40 - displaced by fluid-phase rA-domain, with half-maximal inhibition observed at - 1 mM. EAiC3b did not bind to glutathione-S-transferase (GST) , or to a homologous rA- domain derived from CDlla/CD18. Furthermore, EAiC3b binding to the rCDllb A-domain was blocked by an anti- CDllb mAb that normally blocks EAiC3b binding to cell- bound CDllb/CD18 (CR3). These data establish the specificity of the interaction between the expressed rCDllb A-domain and iC3b. Binding of EAiC3b to the rA-domain is divalent-cation dependent but temperature independent: Binding of CDllb/CD18 (CR3) to EAiC3b in whole cells is absent at 4°C and optimal at 37°C. It also requires the presence of the physiologic divalent cations Mg²⁺ and Ca2+, or Mn²⁺ . The divalent-cation and temperature dependency of EAiC3b binding to rA-domain was therefor measured. Experiment similar to the binding experiments described above demonstrated that divalent cations were essential for binding. One mM MnCl₂, 1 mM MgCl₂ or a combination of l mM MgCl₂ and 0.15 mM CaCl₂ supported this interaction. CaCl₂ alone (0.15 -1 mM) was ineffective. No specific binding was observed if divalent cations were omitted, or when EDTA was included in the reaction mixture. Similarly, a single point mutation (D242A) that impairs the ability of the rA-domain to bind divalent-cations

(Michishita et al., Cell 7:857, 1993) , also impaired its interaction with EAiC3b.

In contrast to the cell-bound heterodimeric receptor, binding of EAiC3b to the rA-domain was temperature-independent. These findings suggest that the temperature dependency of cell-bound CR3 may be required for posttranslational modifications occurring in its cytoplasmic tails, changes in receptor conformation, and/or its cell surface distribution. Binding of A-domain-derived peptides to EAiC3b: In order to further define the region within the A-domain that binds EAiC3b, overlapping synthetic peptides spanning the whole A-domain region of CDllb (Figure 12) , were examined for the ability of each to bind directly to EAiC3b and to inhibit EAiC3b binding to the A-domain. Two overlapping peptides, AM230 and A24 (calculated pi of 10.78 and 3.76 respectively) bound directly to EAiC3b but not to EA, and binding was also visible by the naked eye. AM230 and A24 comprised most of the sequence encoded by exon 8 of the CDllb gene, and had a 14 amino acid overlapping region (Figure 12) . When this region (peptide A7) was synthesized on two separate occasions, adsorbed to plastic and tested, it bound EAiC3b directly, specifically and in a dose-dependent manner. No binding was observed when a scrambled form of A7 was used. Fluid-phase biotinylated A7 also bound directly and specifically to EAiC3b, excluding the possibility that the ligand binding observed with the adsorbed peptide is artifactual in nature.

Whereas the interaction of EAiC3b with the rA- domain was divalent-cation dependent, EAiC3b binding to AM230, A24 and A7 was not significantly altered by removal of divalent cations or by inclusion of EDTA. EAiC3b did not bind to wells coated with A7-derived peptides comprising respectively the N-terminal half (A9) , the C-terminal half (A10) , or the smaller C- terminal peptides B21 and B23. These findings suggest that most of the residues within A7 may be required for iC3b binding. Moreover, microtiter wells precoated with A8, a synthetic peptide from the corresponding A-domain region of CDlla, did not bind to EAiC3b, consistent with the lack of binding of the rCDlla A-domain or of rCDlla/CD18 to EAiC3b. The lack of direct EAiC3b binding by the other CDllb-derived peptides could be caused by differences in the degree of adsorption of peptides to the plastic wells and/or to lower affinities for iC3b. The ability of the purified peptides to bind EAiC3b indirectly was therefor measured. This was done by determining their effect on binding of EAiC3b to immobilized rA-domain. A7 inhibited binding of EAiC3b to the A-domain in a dose-dependent manner, with half-maximal inhibition at 5 mg/ml (- 3.5 mM) . At > 50 mg/ml (35 mM) , A7 inhibited EAiC3b binding to the A-domain completely. This inhibition required the continuous presence of A7, was not secondary to degradation of iC3b or to a toxic effect of the peptide on erythrocytes, since the inhibitory effect was reversible when A7-treated EAiC3b cells were washed prior to their addition to adsorbed rA-domain. The ability of each of the remaining peptides to inhibit EAiC3b-rA-domain interaction was then tested at an approximately three-fold higher peptide concentration (200 mg/ml or 100 mM) . At this concentration, none of the other tested peptides (including the CDlla peptide A8 and Sc. A7) significantly inhibited rCDllb A-domain- binding to EAiC3b.

The ability of A7 to inhibit EAiC3b binding to CR3 (CDllb/CD18) expressed by normal human neutrophils was measured under conditions similar to those used in assessing EAiC3b binding to rA-domain. EAiC3b binding to neutrophils is primarily mediated by CR3, but can also occur in vitro through complement receptor type 1 (CR1) . The effect of A7 on EAiC3b binding was tested in nearly isotonic conditions and in the presence of blocking concentrations of a polyclonal anti-CRl antibody (Ross et al., J . Exp. Med . 158:334, 1983). These experiments demonstrated that EAiC3b binding to adherent neutrophils was primarily CR3 mediated under these conditions, since it was inhibited by the anti-CR3 mAb 903, which inhibits iC3b binding selectively. A7 but not the control A- domain-derived peptide A4, significantly inhibited CR3- dependent binding of EAiC3b to neutrophils with 70% inhibition observed at 100 mM and almost complete inhibition seen at 140 mM. These findings indicate that A7 is the major site in CR3 responsible for its interaction with iC3b. Monoclonal Antibodies Monoclonal antibodies directed against CDll or CD18 can be used to antagonize CD11/CD18-mediated immune response. Useful monoclonal antibodies can be generated by using a peptide of the invention as an immunogen. For example, monoclonal antibodies can be raised against the A domain of CDllb, CDlla or CDllc, or the A domain of any of ,5l-,58.

Anti-CDllb monoclonal antibodies which inhibit iC3b binding (mAb 903) , neutrophil adhesive interactions, e.g., aggregation and chemotaxis, (mAb 904), or both activities (mAb44a) have been identified. Other monoclonal antibodies (OKM-1, which inhibits fibrinogen binding, and OKM9) have also been mapped to this region. Dana et al., J . Immunol . 137:3259 (1986). These monoclonal antibodies recognize epitopes in the A domain of CDllb. Dana et al., JASON 1:549 (1990).

Additional useful monoclonal antibodies can be generated by standard techniques. Preferably, human monoclonal antibodies can be produced. Human monoclonal antibodies can be isolated from a combinatorial library produced by the method of Huse et al. (Science, 246:1275, 1988) . The library can be generated in vivo by immunizing nude or SCID mice whose immune system has been reconstituted with human peripheral blood lymphocytes or spleen cells or in vitro by immunizing human peripheral blood lymphocytes or spleen cells. The immunogen can be any CDllb or CD18 peptide. Similar techniques are described by Duchosal et al., J. Exp. Med. 92:985 (1990) and Mullinax et al., Proc. Nat'l. Acad. USA 87:8095 (1990) . Peptides derived from the A domain of CDlla,

CDllb, or CDllc are preferred immunogens. These peptides can be produced in E. coli transformed by a plasmid encoding all or part of the A domain.

A CD18 peptide can also be used as an immunogen. Three anti-CD18 mAbs with anti-inflammatory properties (TS18, 10F12, 60.3) have been identified. Binding each of these antibodies to CD18 can be abrogated by a specific point mutation within a particular region of CD18 (Asp¹²⁸ to Asn³⁶¹ of Fig. 8) (SEQ ID No.: 45) . Peptide corresponding to this region can be produced in E . coli using a plasmid encoding the A domain. Assays for CDllb for CDllc) peptides. heterodimers and monoclonal antibodies

CDllb (or CDllc) peptides, heterodimers, and monoclonal antibodies such as those described above, can be tested in vitro for inhibition in one of the following five assays: inhibition of granulocyte of phagocyte adhesion to iC3b-coated erythrocytes or bacteria (iC3b binding) , inhibition of phagocytosis, inhibition of monocyte/granulocyte adhesion to endothelium, inhibition of chemotaxis, or inhibition of cell-cell aggregation. These assays can be performed as described in USSN 08/216,081, hereby incorporated by reference. Alternatively, they may be tested in vivo for controlling damage associated with reduced perfusion or immune injury of tissues, as a result of yocardial infarction, burns, frost bite, glomerulonephritis, asthma, adult respiratory distress syndrome, transplant rejection, onset of diabetes mellitus, ischemia, colitis, shock liver syndrome, and resuscitation from hemorrhagic shock. Assays for CDlla peptides. heterodimers and monoclonal antibodies

CDlla peptides, heterodimers and monoclonal antibodies can be tested using the inhibition of endothelial adhesion assay (described above) or a lymphocyte proliferation assay. Arnaout et al., J . Clin . Invest . 74:1291 (1984) describes an assay for inhibition of antigen/mitogen induced lymphocyte proliferation. In Vivo Model for Testing Peptides and Antagonists Damage to tissues injured by ischemia-reperfussion (e.g., heart tissue during myocardial infarction) can be minimized by administering to an animal an inhibitor of CD11/CD18 mediated immune response. A peptide of the invention may be tested for in vivo effectiveness using animals, e.g., dogs, which have been induced to undergo myocardial infarction. See, e.g. Simpson et al. supra . Use

The peptides or monoclonal antibody can be administered intravenously in saline solution generally on the order of mg quantities per 10 kilograms of body weight. The peptide can be administered in combination with other drugs, for example, in combination with, or within six hours to three days after a clot dissolving agent, e.g., tissue plasminogen activator (TPA) , Activase, or Streptokinase.

The screening assays of the invention are useful for identifying potential antagonists (inhibitors) of immune reactions mediated by A-domain containing integrins. Accordingly, the screening methods of the invention are highly useful for limiting the number of candidate antagonists which would otherwise have to be subjected to more complicated screening proceedures involving intact integrin heterodimers or animal models. - 46 -

Other Embodiments

The invention also feature antagonists identified by the screening assays of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: M. Amin Arnaout (ii) TITLE OF INVENTION: METHODS FOR IDENTIFYING INTEGRIN ANTAGONISTS

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(A) APPLICATION NUMBER: PCT/US96/

(B) FILING DATE: 30-JAN-96

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/380,167

(B) FILING DATE: 30-JAN-95

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: John W. Freeman

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(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

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(C) TELEX: 200154

(2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1 Ala Tyr Phe Gly Ala Ser Leu Cys Ser Val Asp Val Asp Ser Asn

5 10 15

Gly Ser Thr Asp Leu Val Leu lie Gly Ala Pro 20 25

(2) INFORMATION FOR SEQ ID NO:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2

Gly Arg Phe Gly Ala Ala Leu Thr Val Leu Gly Asp Val Asn Gly 5 10 15

Asp Lys Leu Thr Asp Val Ala lie Gly Ala Pro 20 25

(2) INFORMATION FOR SEQ ID NO: 3 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3

Gin Tyr Phe Gly Gin Ser Leu Ser Gly Gly Gin Asp Leu Thr Met 5 10 15

Asp Gly Leu Val Asp Leu Thr Val Gly Ala Gin 20 25

(2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4

Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys Pro Leu Pro 5 10 15

Arg Gly Arg Ala Arg Trp Gin Cys Asp Ala Val 20 25 (2) INFORMATION FOR SEQ ID NO: 5 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5

Asp lie Ala Phe Leu lie Asp Gly Ser Gly Ser lie lie Pro His 5 10 15

Asp Phe Arg Arg Met Lys 20

(2) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6

Arg Arg Met Lys Glu Phe Val Ser Thr Val Met Glu Gin Leu Lys 5 10 15

Lys Ser Lys Thr Leu Phe 20

(2) INFORMATION FOR SEQ ID NO: 7 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 7

Ser Leu Met Gin Tyr Ser Glu Glu Phe Arg lie His Phe Thr Phe 5 10 15

Lys Glu Phe Gin Asn Asn 20

(2) INFORMATION FOR SEQ ID NO: 8 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25

(B) TYPE: amino acid (C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8

Pro Asn Pro Arg Ser Leu Val Lys Pro He Thr Gin Leu Leu Gly 5 10 15

Arg Thr His Thr Ala Thr Gly He Arg Lys 20 25

(2) INFORMATION FOR SEQ ID NO: 9 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: amino acid

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(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 9

Arg Lys Val Val Arg Glu Leu Phe Asn He Thr Asn Gly Ala Arg 5 10 15

Lys Asn Ala Phe Lys 20

(2) INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 10

Phe Lys He Leu Val Val He Thr Asp Gly Glu Lys Phe Gly Asp 5 10 15

Pro Leu Gly Tyr Glu Asp Val He Pro Glu Ala Asp Arg 20 25

(2) INFORMATION FOR SEQ ID NO: 11 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear - 51 - (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 11

Arg Glu Gly Val He Arg Tyr Val He Gly Val Gly Asp Ala Phe 5 10 15

Arg Ser Glu Lys Ser Arg 20

(2) INFORMATION FOR SEQ ID NO: 12 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 12

Gin Glu Leu Asn Thr He Ala Ser Lys Pro Pro Arg Asp His Val 5 10 15

Phe Gin Val Asn Asn Phe Glu 20

(2) INFORMATION FOR SEQ ID NO: 13 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19

(B) TYPE: amino acid

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(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 13

Ala Leu Lys Thr He Gin Asn Gin Leu Arg Glu Lys He Phe Ala 5 10 15

He Glu Gly Thr

(2) INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15

(B) TYPE: amino acid

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(D) TOPOLOGY: linear

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Gin Thr Gly Ser Ser Ser Ser Phe Glu His Glu Met Ser Gin Glu - 52 -

(2) INFORMATION FOR SEQ ID NO: 15 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 15

Lys Ser Thr Arg Asp Arg Leu Arg 5

(2) INFORMATION FOR SEQ ID NO: 16 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 16

Phe Arg Ser Glu Lys Ser Arg Gin Glu Leu Asn Thr He Ala Ser 5 10 15

Lys Pro Pro Arg Asp His Val 20

(2) INFORMATION FOR SEQ ID NO: 17 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 17

Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly Tyr Glu Asp Val He 5 10 15

Pro Glu Ala Asp Arg 20

(2) INFORMATION FOR SEQ ID NO: 18 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 (B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

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Lys Glu Phe Gin Asn Asn Pro Asn Pro Arg Ser Leu 5 10

(2) INFORMATION FOR SEQ ID NO: 19 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 19

Gly Thr Gin Thr Gly Ser Ser Ser Ser Phe Glu His Glu Met Ser 5 10 15

Gin Glu Gly

(2) INFORMATION FOR SEQ ID NO: 20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 20

Ser Asn Leu Arg Gin Gin Pro Gin Lys Phe Pro Glu Ala Leu Arg 5 10 15

Gly Cys Pro Gin Glu Asp Ser Asp 20

(2) INFORMATION FOR SEQ ID NO: 21 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 21 Arg Gin Asn Thr Gly Met Trp Glu Ser Asn Ala Asn Val Lys Gly 5 10 15

Thr

(2) INFORMATION FOR SEQ ID NO: 22 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15

(B) TYPE: amino acid

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(D) TOPOLOGY: linear

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Thr Ser Gly Ser Gly He Ser Pro Ser His Ser Gin Arg He Ala

5 10 15

[2) INFORMATION FOR SEQ ID NO: 23 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: amino acid

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(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 23

Asn Gin Arg Gly Ser Leu Tyr Gin Cys Asp Tyr Ser Thr Gly Ser 5 10 15

Cys Glu Pro He Arg 20

(2) INFORMATION FOR SEQ ID NO: 24 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 24

Pro Arg Gly Arg Ala Arg Trp Gin Cys 5

(2) INFORMATION FOR SEQ ID NO: 25 ( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 20

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 25

Lys Leu Ser Pro Arg Leu Gin Tyr Phe Gly Gin Ser Leu Ser Gly 5 10 15

Gly Gin Asp Leu Thr 20

(2) INFORMATION FOR SEQ ID NO: 26 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

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(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 26

Gin Lys Ser Thr Arg Asp Arg Leu Arg Glu Gly Gin 5 10

(2) INFORMATION FOR SEQ ID NO: 27 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 27

Ser Gly Arg Pro His Ser Arg Ala Val Phe Asn Glu Thr Lys Asn 5 10 15

Ser Thr Arg Arg Gin Thr Gin 20

(2) INFORMATION FOR SEQ ID NO: 28 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 28

Cys Glu Thr Leu Lys Leu G n Leu Pro Asn Cys He Glu Asp Pro 5 10 15

Val

(2) INFORMATION FOR SEQ ID NO: 29 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 29

Phe Glu Lys Asn Cys Gly Asn Asp Asn He Cys Gin Asp Asp Leu 5 10 15

(2) INFORMATION FOR SEQ ID NO: 30 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 30

Val Arg Asn Asp Gly Glu Asp Ser Tyr Arg Thr Gin 5 10

(2) INFORMATION FOR SEQ ID NO: 31 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 31

Ser Tyr Arg Lys Val Ser Thr Leu Gin Asn Gin Arg Ser Gin Arg

5 10 15

Ser

(2) INFORMATION FOR SEQ ID NO: 32 - 57 -

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 32

Asp He Ala Phe Leu He Asp Gly Ser

5

(2) INFORMATION FOR SEQ ID NO: 33 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 33

Phe Arg Arg Met Lys Glu Phe Val Ser

5

(2) INFORMATION FOR SEQ ID NO: 34 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 34

Phe Lys He Leu Val Val He Thr Asp Gly Glu 5 10

(2) INFORMATION FOR SEQ ID NO: 35 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 35 - 58 -

Val He Arg Tyr Val He Gly Val Gly Asp Ala 5 10

(2) INFORMATION FOR SEQ ID NO: 36 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 36

Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly 5 10

(2) INFORMATION FOR SEQ ID NO: 37 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 37

Tyr Glu Asp Val He Pro Glu Ala Asp Arg 5 10

(2) INFORMATION FOR SEQ ID NO: 38 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: 1inear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 38

Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Ser Val Cys 5 10 15

Pro Arg Gly Arg Ala Arg Trp Gin Cys Asp Ala Tyr 20 25

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5137 base pairs

(B) TYPE: nucleic acid - 59 -

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

GAATTCCCTC TTTCACCCTG TCTAGGTTGC CAGCAAATCC CACGGGCCTC CTGACGCTGC 60

CCCTGGGGCC ACAGGTCCCT CGAGTGCTGG AAGGATGAAG GATTCCTGCA TCACTGTGAT 120

GGCCATGGCG CTGCTGTCTG GGTTCTTTTT CTTCGCGCCG GCCTCGAGCT ACAACCTGGA 180

CGTGCGGGGC GCGCGGAGCT TCTCCCCACC GCGCGCCGGG AGGCACTTTG GATACCGCGT 240

CCTGCAGGTC GGAAACGGGG TCATCGTGGG AGCTCCAGGG GAGGGGAACA GCACAGGAAG 300

CCTCTATCAG TGCCAGTCGG GCACAGGACA CTGCCTGCCA GTCACCCTGA GAGGTTCCAA 360

CTATACCTCC AAGTACTTGG GCATGACCTT GGCAACAGAC CCCACAGATG GAAGCATTTT 420

GGCCTGTGAC CCTGGGCTGT CTCGAACGTG TGACCAGAAC ACCTATCTGA GTGGCCTGTG 480

TTACCTCTTC CGCCAGAATC TGCAGGGTCC CATGCTGCAG GGGCGCCCTG GTTTTCAGGA 540

ATGTATCAAG GGCAACGTAG ACCTGGTATT TCTGTTTGAT GGTTCGATGA GCTTGCAGCC 600

AGATGAATTT CAGAAAATTC TGGACTTCAT GAAGGATGTG ATGAAGAAAC TCAGCAACAC 660

TTCGTACCAG TTTGCTGCTG TTCAGTTTTC CACAAGCTAC AAAACAGAAT TTGATTTCTC 720

AGATTATGTT AAATGGAAGG ACCCTGATGC TCTGCTGAAG CATGTAAAGC ACATGTTGCT 780

GTTGACAAAT ACCTTTGGTG CCATCAATTA TGTCGCGACA GAGGTGTTCC GGGAGGAGCT 840

GGGGGCCCGG CCAGATGCCA CCAAAGTGCT TATCATCATC ACGGATGGGG AGGCCACTGA 900

CAGTGGCAAC ATCGATGCGG CCAAAGACAT CATCCGCTAC ATCATCGGGA TTGGAAAGCA 960

TTTTCAGACC AAGGAGAGTC AGGAGACCCT CCACAAATTT GCATCAAAAC CCGCGAGCGA 1020

GTTTGTGAAA ATTCTGGACA CATTTGAGAA GCTGAAAGAT CTATTCATCG AGCGGCAGAA 1080

GAAGATCTAT GTCATTGAGG GCACAAGCAA ACAGGACCTG ACTTCCTTCA ACATGGAGCT 1140

GTCCTCCAGC GGCATCAGTG CTGACCTCAG CAGGGGCCAT GCAGTCGTGG GGGCAGTAGG 1200

AGCCAAGGAC TGGGCTGGGG GCTTTCTTGA CCTGAAGGCA GACCTGCAGG ATGACACATT 1260

TATTGGGAAT GAACCATTGA CACCAGAAGT GAGAGCAGGC TATTTGGGTT ACACCGTGAC 1320

CTGGCTGCCC TCCCGGCAAA AGACTTCGTT GCTGGCCTCG GGAGCCCCTC GATACCAGCA 1380

CATGGGCCGA GTGCTGCTGT TCCAAGAGCC ACAGGGCGGA GGACACTGGA GCCAGGTCCA 1440

GACAATCCAT GGGACCCAGA TTGGCTCTTA TTTCGGTGGG GAGCTGTGTG GCGTCGACGT 1500

GGACCAAGAT GGGGAGACAG AGCTGCTGCT GATTGGTGCC CCACTGTTCT ATGGGGAGCA 1560

GAGAGGAGGC CGGGTGTTTA TCTACCAGAG AAGACAGTTG GGGTTTGAAG AAGTCTCAGA 1620 GCTGCAGGGG GACCCCGGCT ACCCACTCGG GCGGTTTGGA GAAGCCATCA CTGCTCTGAC 1680

AGACATCAAC GGCGATGGGC TGGTAGACGT GGCTGTGGGG GCCCCTCTGG AGGAGCAGGG 1740

GGCTGTGTAC ATCTTCAATG GGAGGCACGG GGGGCTTAGT CCCCAGCCAA GTCAGCGGAT 1800

AGAAGGGACC CAAGTGCTCT CAGGAATTCA GTGGTTTGGA CGCTCCATCC ATGGGGTGAA 1860

GGACCTTGAA GGGGATGGCC TGGCAGATGT GGCTGTGGGG GCTGAGAGCC AGATGATCGT 1920

GCTGAGCTCC CGGCCCGTGG TGGATATGGT CACCCTGATG TCCTTCTCTC CAGCTGAGAT 1980

CCCAGTGCAT GAAGTGGAGT CGTCCTATTC AACCAGTAAC AAGATGAAAG AAGGAGTTAA 2040

TATCACAATC TGTTTCCAGA TCAAGTCTCT CTACCCCCAG TTCCAAGGCC GCCTGGTTGC 2100

CAATCTCACT TACACTCTGC AGCTGGATGG CCACCGGACC AGAAGACGGG GGTTGTTCCC 2160

AGGAGGGAGA CATGAACTCA GAAGGAATAT AGCTGTCACC ACCAGCATGT CATGCACTGA 2220

CTTCTCATTT CATTTCCCGG TATGTGTTCA AGACCTCATC TCCCCCATCA ATGTTTCCCT 2280

GAATTTCTCT CTTTGGGAGG AGGAAGGGAC ACCGAGGGAC CAAAGGGCGC AGGGCAAGGA 2340

CATACCGCCC ATCCTGAGAC CCTCCCTGCA CTCGGAAACC TGGGAGATCC CTTTTGAGAA 2400

GAACTGTGGG GAGGACAAGA AGTGTGAGGC AAACTTGAGA GTGTCCTTCT CTCCTGCAAC 2460

ATCCAGAGCC CTGCGTCTAA CTGCTTTTGC CAGCCTCTCT GTGGAGCTGA GCCTGAGTAA 2520

CTTGGAAGAA GATGCTTACT GGGTCCAGCT GGACCTGCAC TTCCCCCCGG GACTCTCCTT 2580

CCGCAAGGTG GAGATGCTGA AGCCCCATAG CCAGATACCT GTGAGCTGCG AGGAGCTTCC 2640

TGAAGAGTCC AGGCTTCTGT CCAGGGCATT ATCTTGCAAT GTGAGCTCTC CCATCTTCAA 2700

AGCAGGCCAC TCGGTTGCTC TGCAGATGAT GTTTAATACA CTGGTAAACA GCTCCTGGGG 2760 GGACTCGGTT GAATTGCACG CCAATGTGAC CTGTAACAAT GAGGACTCAG ACCTCCTGGA .2820

GGACAACTCA GCCACTACCA TCATCCCCAT CCTGTACCCC ATCAACATCC TCATCCAGGA 2880

CCAAGAAGAC TCCACACTCT ATGTCAGTTT CACCCCCAAA GGCCCCAAGA TCCACCAAGT 2940

CAAGCACATG TACCAGGTGA GGATCCAGCC TTCCATCCAC GACCACAACA TACCCACCCT 3000

GGAGGCTGTG GTTGGGGTGC CACAGCCTCC CAGCGAGGGG CCCATCACAC ACCAGTGGAG 3060

CGTGCAGATG GAGCCTCCCG TGCCCTGCCA CTATGAGGAT CTGGAGAGGC TCCCGGATGC 3120

AGCTGAGCCT TGTCTCCCCG GACCCCTGTT CCGCTGCCCT GTTGTCTTCA GGCAGGAGAT 3180

CCTCGTCCAA GTGATCGGGA CTCTGGAGCT GGTGGGAGAG ATCGAGGCCT CTTCCATGTT 3240

CAGCCTCTGC AGCTCCCTCT CCATCTCCTT CAACAGCAGC AAGCATTTCC ACCTCTATGG 3300

CAGCAACGCC TCCCTGGCCC AGGTTGTCAT GAAGGTTGAC GTGGTGTATG AGAAGCAGAT 3360

GCTCTACCTC TACGTGCTGA GCGGCATCGG GGGGCTGCTG CTGCTGCTGC TCATTTCATA 3420

GTGCTGTACA AGGTTGGTTT CTTCAAACGG AACCTGAAGG AGAAGATGGA GGCTGGCAGA 3480

GGTGTCCCGA ATGGAATCCC TGCAGAAGAC TCTGAGCAGC TGGCATCTGG GCAAGAGGCT 3540 GGGGATCCCG GCTGCCTGAA GCCCCTCCAT GAGAAGGACT CTGAGAGTGG TGGTGGCAAG 3600

GACTGAGTCC AGCCTGTGAG GTGCAGAGTG CCCAGAACTG GACTCAGGAT GCCCAGGGCC 3660

ACTTCGCCTC TGCCTGCATT CTGCCGTGTG CCCTCGGGCG AGTCACTGCC TCTCCCTGGC 3720

CCTCAGTTTC CCTATCTCGA ACATGGAACT CATTCCTGAA TGTCTCCTTT GCAGGCTCAT 3780

AGGGAAGACC TGCTGAGGGA CCAGCCAAGA GGGCTGCAAA AGTGAGGGCT TGTCATTACC 3840

AGACGGTTCA CCAGCCTCTC TTGGTTCCTT CCTTGGAAGA GAATGTCTGA TCTAAATGTG 3900

GAGAAACTGT AGTCTCAGGA CCTAGGGATG TTCTGGCCCT CACCCCTGCC CTGGGATGTC 3960

CACAGATGCC TCCACCCCCC AGAACCTGTC CTTGCACACT CCCCTGCACT GGAGTCCAGT 4020

CTCTTCTGTT GGCAGAAAGC AAATGTGACC TGTGTCACTA CGTGACTGTG GCACACGCCT 4080

TGTTCTTGGC CAAAGACCAA ATTCCTTGGC ATGCCTTCCA GCACCCTGCA AAATGAGACC 4140

CTCGTGGCCT TCCCCAGCCT CTTCTAGAGC CGTGATGCCT CCCTGTTGAA GCTCTGGTGA 4200

CACCAGCCTT TCTCCCAGGC CAGGCTCCTT CCTGTCTTCC TGCATTCACC CAGACAGCTC 4260

CCTCTGCCTG AACCTTCCAT CTCGCCCACC CCTCCTTCCT TGACCAGCAG ATCCCAGCTC 4320

ACGTCACACA CTTGGTTGGG TCCTCACATC TTTCACACTT CCACCACCCT GCACTACTCC 4380

CTCAAAGCAC ACGTCATGTT TCTTCATCCG GCAGCCTGGA TGTTTTTTCC CTGTTTAATG 4440

ATTGACGTAC TTAGCAGCTA TCTCTCAGTG AACTGTGAGG GTAAAGGCTA TACTTGTCTT 4500

GTTCACCTTG GGATGACGCC GCATGATATG TCAGGGCGTG GGACATCTAG TAGGTGCTTG 4560

ACATAATTTC ACTGAATTAA TGACAGAGCC AGTGGGAAGA TACAGAAAAA GAGGGCCGGG 4620

GCTGGGCGCG GTGGTTCACG CCTGTAATCC CAGCACTTTG GGAGGCCAAG GAGGGTGGAT 4680

CACCTGAGGT CAGGAGTTAG AGGCCAGCCT GGCGAAACCC CATCTCTACT AAAAATACAA 4740

AATCCAGGCG TGGTGGCACA CACCTGTAGT CCCAGCTACT CAGGAGGTTG AGGTAGGAGA 4800

ATTGCTTGAA CCTGGGAGGT GGAGGTTGCA GTGAGCCAAG ATTGCGCCAT TGCACTCCAG 4860

CCTGGGCAAC ACAGCGAGAC TCCGTCTCAA GGAAAAAATA AAAATAAAAA GCGGGCACGG 4920

GCCCGGACAT CCCCACCCTT GGAGGCTGTC TTCTCAGGCT CTGCCCTGCC CTAGCTCCAC 4980

ACCCTCTCCC AGGACCCATC ACGCCTGTGC AGTGGCCCCC ACAGAAAGAC TGAGCTCAAG 5040

GTGGGAACCA CGTCTGCTAA CTTGGAGCCC CAGTGCCAAG CACAGTGCCT GCATGTATTT 5100

ATCCAATAAA TGTGAAATTC TGTCCAAAAA AAAAAAA 5137

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3533 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

TGGCTTCCTT GTGGTTCCTC AGTGGTGCCT GCAACCCCTG GTTCACCTCC TTCCAGGTTC 60

TGGCCCTTCC AGCCATGGCT CTCAGAGTCC TTCTGTTAAC AGCCTTGACC TTATGTCATG 120

GGTTCAACTT GGACACTGAA AACGCAATGA CCTTCCAAGA GAACGCAAGG GGCTTCGGGC 180

AGAGCGTGGT CCAGCTTCAG GGATCCAGGG TGGTGGTTGG AGCCCCCCAG GAGATAGTGG 240

CTGCCAACCA AAGGGGCAGC CTCTACCAGT GCGACTACAG CACAGGCTCA TGCGAGCCCA 300

TCCGCCTGCA GGTCCCCGTG GAGGCCGTGA ACATGTCCCT GGGCCTGTCC CTGGCAGCCA 360

CCACCAGCCC CCCTCAGCTG CTGGCCTGTG GTCCCACCGT GCACCAGACT TGCAGTGAGA 420

ACACGTATGT GAAAGGGCTC TGCTTCCTGT TTGGATCCAA CCTACGGCAG CAGCCCCAGA 480

AGTTCCCAGA GGCCCTCCGA GGGTGTCCTC AAGAGGATAG TGACATTGCC TTCTTGATTG 540

ATGGCTCTGG TAGCATCATC CCACATGACT TTCGGCGGAT GAAGGAGTTT GTCTCAACTG 600

TGATGGAGCA ATTAAAAAAG TCCAAAACCT TGTTCTCTTT GATGCAGTAC TCTGAAGAAT 660

TCCGGATTCA CTTTACCTTC AAAGAGTTCC AGAACAACCC TAACCCAAGA TCACTGGTGA 720

AGCCAATAAC GCAGCTGCTT GGGCGGACAC ACACGGCCAC GGGCATCCGC AAAGTGGTAC 780

GAGAGCTGTT TAACATCACC AACGGAGCCC GAAAGAATGC CTTTAAGATC CTAGTTGTCA 840

TCACGGATGG AGAAAAGTTT GGCGATCCCT TGGGATATGA GGATGTCATC CCTGAGGCAG 900

ACAGAGAGGG AGTCATTCGC TACGTCATTG GGGTGGGAGA TGCCTTCCGC AGTGAGAAAT 960

CCCGCCAAGA GCTTAATACC ATCGCATCCA AGCCGCCTCG TGATCACGTG TTCCAGGTGA 1020

ATAACTTTGA GGCTCTGAAG ACCATTCAGA ACCAGCTTCG GGAGAAGATC TTTGCGATCG 1080

AGGGTACTCA GACAGGAAGT AGCAGCTCCT TTGAGCATGA GATGTCTCAG GAAGGCTTCA 1140

GCGCTGCCAT CACCTCTAAT GGCCCCTTGC TGAGCACTGT GGGGAGCTAT GACTGGGCTG 1200

GTGGAGTCTT TCTATATACA TCAAAGGAGA AAAGCACCTT CATCAACATG ACCAGAGTGG 1260

ATTCAGACAT GAATGATGCT TACTTGGGTT ATGCTGCCGC CATCATCTTA CGGAACCGGG 1320

TGCAAAGCCT GGTTCTGGGG GCACCTCGAT ATCAGCACAT CGGCCTGGTA GCGATGTTCA 1380

GGCAGAACAC TGGCATGTGG GAGTCCAACG CTAATGTCAA GGGCACCCAG ATCGGCGCCT 1440

ACTTCGGGGC CTCCCTCTGC TCCGTGGACG TGGACAGCAA CGGCAGCACC GACCTGGTCC 1500

TCATCGGGGC CCCCCATTAC TACGAGCAGA CCCGAGGGGG CCAGGTGTCC GTGTGCCCCT 1560

TGCCCAGGGG GAGGGCTCGG TGGCAGTGTG ATGCTGTTCT CTACGGGGAG CAGGGCCAAC 1620

CCTGGGGCCG CTTTGGGGCA GCCCTAACAG TGCTGGGGGA CGTAAATGGG GACAAGCTGA 1680 - 63 -

CGGACGTGGC CATTGGGGCC CCAGGAGAGG AGGACAACCG GGGTGCTGTT TACCTGTTTC 1740

ACGGAACCTC AGGATCTGGC ATCAGCCCCT CCCATAGCCA GCGGATAGCA GGCTCCAAGC 1800

TCTCTCCCAG GCTCCAGTAT TTTGGTCAGT CACTGAGTGG GGGCCAGGAC CTCACAATGG 1860

ATGGACTGGT AGACCTGACT GTAGGAGCCC AGGGGCACGT GCTGCTGCTC AGGTCCCAGC 1920

CAGTACTGAG AGTCAAGGCA ATCATGGAGT TCAATCCCAG GGAAGTGGCA AGGAATGTAT 1980

TTGAGTGTAA TGATCAAGTG GTGAAAGGCA AGGAAGCCGG AGAGGTCAGA GTCTGCCTCC 2040

ATGTCCAGAA GAGCACACGG GATCGGCTAA GAGAAGGACA GATCCAGAGT GTTGTGACTT 2100

ATGACCTGGC TCTGGACTCC GGCCGCCCAC ATTCCCGCGC CGTCTTCAAT GAGACAAAGA 2160

ACAGCACACG CAGACAGACA CAGGTCTTGG GGCTGACCCA GACTTGTGAG ACCCTGAAAC 2220

TACAGTTGCC GAATTGCATC GAGGACCCAG TGAGCCCCAT TGTGCTGCGC CTGAACTTCT 2280

CTCTGGTGGG AACGCCATTG TCTGCTTTCG GGAACCTCCG GCCAGTGCTG GCGGAGGATG 2340

CTCAGAGACT CTTCACAGCC TTGTTTCCCT TTGAGAAGAA TTGTGGCAAT GACAACATCT 2400

GCCAGGATGA CCTCAGCATC ACCTTCAGTT TCATGAGCCT GGACTGCCTC GTGGTGGGTG 2460

GGCCCCGGGA GTCTAACGTG ACAGTGACTG TGAGAAATGA TGGTGAGGAC TCCTACAGGA 2520

CACAGGTCAC CTTCTTCTTC CCGCTTGACC TGTCCTACCG GAAGGTGTCC ACACTCCAGA 2580

ACCAGCGCTC ACAGCGATCC TGGCGCCTGG CCTGTGAGTC TGCCTCCTCC ACCGAAGTGT 2640

CTGGGGCCTT GAAGAGCACC AGCTGCAGCA TAAACCACCC CATCTTCCCG GAAAACTCAG 2700

AGGTCACCTT TAATATCACG TTTGATGTAG ACTCTAAGGC TTCCCTTGGA AACAAACTGC 2760

TCCTCAAGGC CAATGTGACC AGTGAGAACA ACATGCCCAG AACCAACAAA ACCGAATTCC 2820

AACTGGAGCT GCCGGTGAAA TATGCTGTCT ACATGGTGGT CACCAGCCAT GGGGTCTCCA 2880

CTAAATATCT CAACTTCACG GCCTCAGAGA ATACCAGTCG GGTCATGCAG CATCAATATC 2940

AGGTCAGCAA CCTGGGGCAG AGGAGCCCCC CCATCAGCCT GGTGTTCTTG GTGCCCGTCC 3000

GGCTGAACCA GACTGTCATA TGGGACCGCC CCCAGGTCAC CTTCTCCGAG AACCTCTCGA 3060

GTACGTGCCA CACCAAGGAG CGCTTGCCCT CTCACTCCGA CTTTCTGGCT GAGCTTCGGA 3120

AGGCCCCCGT GGTGAACTGC TCCATCGCTG TCTGCCAGAG AATCCAGTGT GACATCCCGT 3180

TCTTTGGCAT CCAGGAAGAA TTCAATGCTA CCCTCAAAGG CAACCTCTCG TTTGACTGGT 3240

ACATCAAGAC CTCGCATAAC CACCTCCTGA TCGTGAGCAC AGCTGAGATC TTGTTTAACG 3300

ATTCCGTGTT CACCCTGCTG CCGGGACAGG GGGCGTTTGT GAGGTCCCAG ACGGAGACCA 3360

AAGTGGAGCC GTTCGAGGTC CCCAACCCCC TGCCGCTCAT CGTGGGCAGC TCTGTCGGGG 3420

GACTGCTGCT CCTGGCCCTC ATCACCGCCG CGCTGTACAA GCTCGGCTTC TTCAAGCGGC 3480

AATACAAGGA CATGATGAGT GAAGGGGGTC CCCCGGGGGC CGAACCCCAG TAG 3533 (2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2310 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

ATGCTGGGCC TGCGCCCCCC ACTTCTCGCC CTGGTGGGGC TGCTCTCCCT CGGGTGCGTC 60

CTCTCTCAGG AGTGCACGAA GTTCAAGGTC AGCAGCTGCC GGGAATGCAT CGAGTCGGGG 120

CCCGGCTGCA CCTGGTGCCA GAAGCTGAAC TTCACAGGGC CGGGGGATCC TGACTCCATT 180

CGCTGCGACA CCCGGCCACA GCTGCTCATG AGGGGCTGTG CGGCTGACGA CATCATGGAC 240

CCCACAAGCC TCGCTGAAAC CCAGGAAGAC CACAATGGGG GCCAGAAGCA GCTGTCCCCA 300

CAAAAAGTGA CGCTTTACCT GCGACCAGGC CAGGCAGCAG CGTTCAACGT GACCTTCCGG 360

CGGGCCAAGG GCTACCCCAT CGACCTGTAC TATCTGATGG ACCTCTCCTA CTCCATGCTT 420

GATGACCTCA GGAATGTCAA GAAGCTAGGT GGCGACCTGC TCCGGGCCCT CAACGAGATC 480

ACCGAGTCCG GCCGCATTGG CTTCGGGTCC TTCGTGGACA AGACCGTGCT GCCGTTCGTG 540

AACACGCACC CTGATAAGCT GCGAAACCCA TGCCCCAACA AGGAGAAAGA GTGCCAGCCC 600

CCGTTTGCCT TCAGGCACGT GCTGAAGCTG ACCAACAACT CCAACCAGTT TCAGACCGAG 660

GTCGGGAAGC AGCTGATTTC CGGAAACCTG GATGCACCCG AGGGTGGGCT GGACGCCATG 720

ATGCAGGTCG CCGCCTGCCC GGAGGAAATC GGCTGGCGCA ACGTCACGCG GCTGCTGGTG 780

TTTGCCACTG ATGACGGCTT CCATTTCGCG GGCGACGGAA AGCTGGGCGC CATCCTGACC 840

CCCAACGACG GCCGCTGTCA CCTGGAGGAC AACTTGTACA AGAGGAGCAA CGAATTCGAC 900

TACCCATCGG TGGGCCAGCT GGCGCACAAG CTGGCTGAAA ACAACATCCA GCCCATCTTC 960

GCGGTGACCA GTAGGATGGT GAAGACCTAC GAGAAACTCA CCGAGATCAT CCCCAAGTCA 1020

GCCGTGGGGG AGCTGTCTGA GGACTCCAGC AATGTGGTCC ATCTCATTAA GAATGCTTAC 1080

AATAAACTCT CCTCCAGGGT CTTCCTGGAT CACAACGCCC TCCCCGACAC CCTGAAAGTC 1140

ACCTACGACT CCTTCTGCAG CAATGGAGTG ACGCACAGGA ACCAGCCCAG AGGTGACTGT 1200

GATGGCGTGC AGATCAATGT CCCGATCACC TTCCAGGTGA AGGTCACGGC CACAGAGTGC 1260

ATCCAGGAGC AGTCGTTTGT CATCCGGGCG CTGGGCTTCA CGGACATAGT GACCGTGCAG 1320

GTTCTTCCCC AGTGTGAGTG CCGGTGCCGG GACCAGAGCA GAGACCGCAG CCTCTGCCAT 1380

GGCAAGGGCT TCTTGGAGTG CGGCATCTGC AGGTGTGACA CTGGCTACAT TGGGAAAAAC 1440 TGTGAGTGCC AGACACAGGG CCGGAGCAGC CAGGAGCTGG AAGGAAGCTG CCGGAAGGAC 1500

AACAACTCCA TCATCTGCTC AGGGCTGGGG GACTGTGTCT GCGGGCAGTG CCTGTGCCAC 1560

ACCAGCGACG TCCCCGGCAA GCTGATATAC GGGCAGTACT GCGAGTGTGA CACCATCAAC 1620

TGTGAGCGCT ACAACGGCCA GGTCTGCGGC GGCCCGGGGA GGGGGCTCTG CTTCTGCGGG 1680

AAGTGCCGCT GCCACCCGGG CTTTGAGGGC TCAGCGTGCC AGTGCGAGAG GACCACTGAG 1740

GGCTGCCTGA ACCCGCGGCG TGTTGAGTGT AGTGGTCGTG GCCGGTGCCG CTGCAACGTA 1800

TGCGAGTGCC ATTCAGGCTA CCAGCTGCCT CTGTGCCAGG AGTGCCCCGG CTGCCCCTCA 1860

CCCTGTGGCA AGTACATCTC CTGCGCCGAG TGCCTGAAGT TCGAAAAGGG CCCCTTTGGG 1920

AAGAACTGCA GCGCGGCGTG TCCGGGCCTG CAGCTGTCGA ACAACCCCGT GAAGGGCAGG 1980

ACCTGCAAGG AGAGGGACTC AGAGGGCTGC TGGGTGGCCT ACACGCTGGA GCAGCAGGAC 2040

GGGATGGACC GCTACCTCAT CTATGTGGAT GAGAGCCGAG AGTGTGTGGC AGGCCCCAAC 2100

ATCGCCGCCA TCGTCGGGGG CACCGTGGCA GGCATCGTGC TGATCGGCAT TCTCCTGCTG 2160

GTCATCTGGA AGGCTCTGAT CCACCTGAGC GACCTCCGGG AGTACAGGCG CTTTGAGAAG 2220

GAGAAGCTCA AGTCCCAGTG GAACAATGAT AATCCCCTTT TCAAGAGCGC CACCACGACG 2280

GTCATGAACC CCAAGTTTGC TGAGAGTTAG 2310 (2) INFORMATION FOR SEQ ID NO: 42 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1170

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 42

Met Lys Asp Ser Cys He Thr Val Met Ala Met Ala Leu Leu Ser 5 10 15

Gly Phe Phe Phe Phe Ala Pro Ala Ser Ser Tyr Asn Leu Asp Val 20 25 30

Arg Gly Ala Arg Ser Phe Ser Pro Pro Arg Ala Gly Arg His Phe 35 40 50

Gly Tyr Arg Val Leu Gin Val Gly Asn Gly Val He Val Gly Ala 55 60 65

Pro Gly Glu Gly Asn Ser Thr Gly Ser Leu Tyr Gin Cys Gin Ser 70 75 80

Gly Thr Gly His Cys Leu Pro Val Thr Leu Arg Gly Ser Asn Tyr 85 90 95

Thr Ser Lys Tyr Leu Gly Met Thr Leu Ala Thr Asp Pro Thr Asp 100 105 115 Gly Ser He Leu Ala Cys Asp Pro Gly Leu Ser Arg Thr Cys Asp

120 125 130

Gin Asn Thr Tyr Leu Ser Gly Leu Cys Tyr Leu Phe Arg Gin Asn 135 140 145

Leu Gin Gly Pro Met Leu Gin Gly Arg Pro Gly Phe Gin Glu Cys 150 155 160

He Lys Gly Asn Val Asp Leu Val Phe Leu Phe Asp Gly Ser Met 165 170 175

Ser Leu Gin Pro Asp Glu Phe Gin Lys He Leu Asp Phe Met Lys 180 185 190

Asp Val Met Lys Lys Leu Ser Asn Thr Ser Tyr Gin Phe Ala Ala 195 200 205

Val Gin Phe Ser Thr Ser Tyr Lys Thr Glu Phe Asp Phe Ser Asp 215 220 225

Tyr Val Lys Trp Lys Asp Pro Asp Ala Leu Leu Lys His Val Lys 230 235 240

His Met Leu Leu Leu Thr Asn Thr Phe Gly Ala He Asn Tyr Val 245 250 255

Ala Thr Glu Val Phe Arg Glu Glu Leu Gly Ala Arg Pro Asp Ala 260 265 270

Thr Lys Val Leu He He He Thr Asp Gly Glu Ala Thr Asp Ser 275 280 285

Gly Asn He Asp Ala Ala Lys Asp He He Arg Tyr He He Gly 290 295 300

He Gly Lys His Phe Gin Thr Lys Glu Ser Gin Glu Thr Leu His 305 310 315

Lys Phe Ala Ser Lys Pro Ala Ser Glu Phe Val Lys He Leu Asp 320 325 330

Thr Phe Glu Lys Leu Lys Asp Leu Phe He Glu Arg Gin Lys Lys 335 340 345

He Tyr Val He Glu Gly Thr Ser Lys Gin Asp Leu Thr Ser Phe 350 355 360

Asn Met Glu Leu Ser Ser Ser Gly He Ser Ala Asp Leu Ser Arg 365 370 375

Gly His Ala Val Val Gly Ala Val Gly Ala Lys Asp Trp Ala Gly 380 385 390

Gly Phe Leu Asp Leu Lys Ala Asp Leu Gin Asp Asp Thr Phe He 395 400 405

Gly Asn Glu Pro Leu Thr Pro Glu Val Arg Ala Gly Tyr Leu Gly 415 420 425

Tyr Thr Val Thr Trp Leu Pro Ser Arg Gin Lys Thr Ser Leu Leu 430 435 440 Ala Ser Gly Ala Pro Arg Tyr Gin His Met Gly Arg Val Leu Leu 445 450 455

Phe Gin Glu Pro Gin Gly Gly Gly His Trp Ser Gin Val Gin Thr 460 465 470

He His Gly Thr Gin He Gly Ser Tyr Phe Gly Gly Glu Leu Cys 475 480 485

Gly Val Asp Val Asp Gin Asp Gly Glu Thr Glu Leu Leu Leu He 490 495 500

Gly Ala Pro Leu Phe Tyr Gly Glu Gin Arg Gly Gly Arg Val Phe 505 510 515

He Tyr Gin Arg Arg Gin Leu Gly Phe Glu Glu Val Ser Glu Leu 520 525 530

Gin Gly Asp Pro Gly Tyr Pro Leu Gly Arg Phe Gly Glu Ala He 535 540 545

Thr Ala Leu Thr Asp He Asn Gly Asp Gly Leu Val Asp Val Ala 550 555 560

Val Gly Ala Pro Leu Glu Glu Gin Gly Ala Val Tyr He Phe Asn 565 570 575

Gly Arg His Gly Gly Leu Ser Pro Gin Pro Ser Gin Arg He Glu 580 585 590

Gly Thr Gin Val Leu Ser Gly He Gin Trp Phe Gly Arg Ser He 595 600 605

His Gly Val Lys Asp Leu Glu Gly Asp Gly Leu Ala Asp Val Ala 610 615 620

Val Gly Ala Glu Ser Gin Met He Val Leu Ser Ser Arg Pro Val 625 630 635

Val Asp Met Val Thr Leu Met Ser Phe Ser Pro Ala Glu He Pro 640 645 650

Val His Glu Val Glu Ser Ser Tyr Ser Thr Ser Asn Lys Met Lys 655 670 675

Glu Gly Val Asn He Thr He Cys Phe Gin He Lys Ser Leu Tyr 680 685 690

Pro Gin Phe Gin Gly Arg Leu Val Ala Asn Leu Thr Tyr Thr Leu 695 670 675

Gin Leu Asp Gly His Arg Thr Arg Arg Arg Gly Leu Phe Pro Gly 680 685 690

Gly Arg His Glu Leu Arg Arg Asn He Ala Val Thr Thr Ser Met 695 700 705

Ser Cys Thr Asp Phe Ser Phe His Phe Pro Val Cys Val Gin Asp 710 715 720

Leu He Ser Pro He Asn Val Ser Leu Asn Phe Ser Leu Trp Glu 725 730 735 Glu Glu Gly Thr Pro Arg Asp Gin Arg Ala Gin Gly Lys Asp He 740 745 750

Pro Pro He Leu Arg Pro Ser Leu His Ser Glu Thr Trp Glu He 755 760 765

Pro Phe Glu Lys Asn Cys Gly Glu Asp Lys Lys Cys Glu Ala Asn 770 775 780

Leu Arg Val Ser Phe Ser Pro Ala Thr Ser Arg Ala Leu Arg Leu 785 790 795

Thr Ala Phe Ala Ser Leu Ser Val Glu Leu Ser Leu Ser Asn Leu 800 805 810

Glu Glu Asp Ala Tyr Trp Val Gin Leu Asp Leu His Phe Pro Pro 815 820 825

Gly Leu Ser Phe Arg Lys Val Glu Met Leu Lys Pro His Ser Gin 830 835 840

He Pro Val Ser Cys Glu Glu Leu Pro Glu Glu Ser Arg Leu Leu 845 850 855

Ser Arg Ala Leu Ser Cys Asn Val Ser Ser Pro He Phe Lys Ala 860 865 870

Gly His Ser Val Ala Leu Gin Met Met Phe Asn Thr Leu Val Asn 875 880 885

Ser Ser Trp Gly Asp Ser Val Glu Leu His Ala Asn Val Thr Cys 890 895 900

Asn Asn Glu Asp Ser Asp Leu Leu Glu Asp Asn Ser Ala Thr Thr 905 910 915

He He Pro He Leu Tyr Pro He Asn He Leu He Gin Asp Gin 920 925 930

Glu Asp Ser Thr Leu Tyr Val Ser Phe Thr Pro Lys Gly Pro Lys 935 940 945

He His Gin Val Lys His Met Tyr Gin Val Arg He Gin Pro Ser 950 955 960

He His Asp His Asn He Pro Thr Leu Glu Ala Val Val Gly Val 965 970 975

Pro Gin Pro Pro Ser Glu Gly Pro He Thr His Gin Trp Ser Val 980 985 990

Gin Met Glu Pro Pro Val Pro Cys His Tyr Glu Asp Leu Glu Arg 995 1000 1005

Leu Pro Asp Ala Ala Glu Pro Cys Leu Pro Gly Pro Leu Phe Arg 1010 1015 • 1020

Cys Pro Val Val Phe Arg Gin Glu He Leu Val Gin Val He Gly 1025 1030 1035

Thr Leu Glu Leu Val Gly Glu He Glu Ala Ser Ser Met Phe Ser 1040 1045 1050 Leu Cys Ser Ser Leu Ser He Ser Phe Asn Ser Ser Lys His Phe 1055 1060 1065

His Leu Tyr Gly Ser Asn Ala Ser Leu Ala Gin Val Val Met Lys 1070 1075 1080

Val Asp Val Val Tyr Glu Lys Gin Met Leu Tyr Leu Tyr Val Leu 1085 1090 1095

Ser Gly He Gly Gly Leu Leu Leu Leu Leu Leu He Xaa He Val 1100 1105 1110

Leu Tyr Lys Val Gly Phe Phe Lys Arg Asn Leu Lys Glu Lys Met 1115 1120 1125

Glu Ala Gly Arg Gly Val Pro Asn Gly He Pro Ala Glu Asp Ser 1130 1135 1140

Glu Gin Leu Ala Ser Gly Gin Glu Ala Gly Asp Pro Gly Cys Leu 1145 1150 1155

Lys Pro Leu His Glu Lys Asp Ser Glu Ser Gly Gly Gly Lys Asp 1160 1165 1170

(2) INFORMATION FOR SEQ ID NO: 43 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1152

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 43

Met Ala Leu Arg Val Leu Leu Leu Thr Ala Leu Thr Leu Cys His 5 10 15

Gly Phe Asn Leu Asp Thr Glu Asn Ala Met Thr Phe Gin Glu Asn 20 25 30

Ala Arg Gly Phe Gly Gin Ser Val Val Gin Leu Gin Gly Ser Arg 35 40 50

Val Val Val Gly Ala Pro Gin Glu He Val Ala Ala Asn Gin Arg 55 60 65

Gly Ser Leu Tyr Gin Cys Asp Tyr Ser Thr Gly Ser Cys Glu Pro 70 75 80

He Arg Leu Gin Val Pro Val Glu Ala Val Asn Met Ser Leu Gly 85 90 95

Leu Ser Leu Ala Ala Thr Thr Ser Pro Pro Gin Leu Leu Ala Cys 100 105 115

Gly Pro Thr Val His Gin Thr Cys Ser Glu Asn Thr Tyr Val Lys 120 125 130

Gly Leu Cys Phe Leu Phe Gly Ser Asn Leu Arg Gin Gin Pro Gin 135 140 145

Lys Phe Pro Glu Ala Leu Arg Gly Cys Pro Gin Glu Asp Ser Asp 150 155 160

He Ala Phe Leu He Asp Gly Ser Gly Ser He He Pro His Asp 165 170 175

Phe Arg Arg Met Lys Glu Phe Val Ser Thr Val Met Glu Gin Leu 180 185 190

Lys Lys Ser Lys Thr Leu Phe Ser Leu Met Gin Tyr Ser Glu Glu 195 200 205

Phe Arg He His Phe Thr Phe Lys Glu Phe Gin Asn Asn Pro Asn 215 220 225

Pro Arg Ser Leu Val Lys Pro He Thr Gin Leu Leu Gly Arg Thr 230 235 240

His Thr Ala Thr Gly He Arg Lys Val Val Arg Glu Leu Phe Asn 245 250 255

He Thr Asn Gly Ala Arg Lys Asn Ala Phe Lys He Leu Val Val 260 265 270

He Thr Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly Tyr Glu Asp 275 280 285

Val He Pro Glu Ala Asp Arg Glu Gly Val He Arg Tyr Val He 290 295 300

Gly Val Gly Asp Ala Phe Arg Ser Glu Lys Ser Arg Gin Glu Leu 305 310 315

Asn Thr He Ala Ser Lys Pro Pro Arg Asp His Val Phe Gin Val 320 325 330

Asn Asn Phe Glu Ala Leu Lys Thr He Gin Asn Gin Leu Arg Glu 335 340 345

Lys He Phe Ala He Glu Gly Thr Gin Thr Gly Ser Ser Ser Ser 350 355 360

Phe Glu His Glu Met Ser Gin Glu Gly Phe Ser Ala Ala He Thr 365 370 375

Ser Asn Gly Pro Leu Leu Ser Thr Val Gly Ser Tyr Asp Trp Ala 380 385 390

Gly Gly Val Phe Leu Tyr Thr Ser Lys Glu Lys Ser Thr Phe He 395 400 405

Asn Met Thr Arg Val Asp Ser Asp Met Asn Asp Ala Tyr Leu Gly 415 420 425

Tyr Ala Ala Ala He He Leu Arg Asn Arg Val Gin Ser Leu Val 430 435 440

Leu Gly Ala Pro Arg Tyr Gin His He Gly Leu Val Ala Met Phe 445 450 455 Arg Gin Asn Thr Gly Met Trp Glu Ser Asn Ala Asn Val Lys Gly 460 465 470

Thr Gin He Gly Ala Tyr Phe Gly Ala Ser Leu Cys Ser Val Asp 475 480 485

Val Asp Ser Asn Gly Ser Thr Asp Leu Val Leu He Gly Ala Pro 490 495 500

His Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys Pro 505 510 515

Leu Pro Arg Gly Arg Ala Arg Trp Gin Cys Asp Ala Val Leu Tyr 520 525 530

Gly Glu Gin Gly Gin Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr 535 540 545

Val Leu Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Ala He 550 555 560

Gly Ala Pro Gly Glu Glu Asp Asn Arg Gly Ala Val Tyr Leu Phe 565 570 575

His Gly Thr Ser Gly Ser Gly He Ser Pro Ser His Ser Gin Arg 580 585 590

He Ala Gly Ser Lys Leu Ser Pro Arg Leu Gin Tyr Phe Gly Gin 595 600 605

Ser Leu Ser Gly Gly Gin Asp Leu Thr Met Asp Gly Leu Val Asp 610 615 620

Leu Thr Val Gly Ala Gin Gly His Val Leu Leu Leu Arg Ser Gin 625 630 635

Pro Val Leu Arg Val Lys Ala He Met Glu Phe Asn Pro Arg Glu 640 645 650

Val Ala Arg Asn Val Phe Glu Cys Asn Asp Gin Val Val Lys Gly 655 670 675

Lys Glu Ala Gly Glu Val Arg Val Cys Leu His Val Gin Lys Ser 680 685 690

Thr Arg Asp Arg Leu Arg Glu Gly Gin He Gin Ser Val Val Thr 695 670 675

Tyr Asp Leu Ala Leu Asp Ser Gly Arg Pro His Ser Arg Ala Val 680 685 690

Phe Asn Glu Thr Lys Asn Ser Thr Arg Arg Gin Thr Gin Val Leu 695 700 705

Gly Leu Thr Gin Thr Cys Glu Thr Leu Lys Leu Gin Leu Pro Asn 710 715 720

Cys He Glu Asp Pro Val Ser Pro He Val Leu Arg Leu Asn Phe 725 730 735

Ser Leu Val Gly Thr Pro Leu Ser Ala Phe Gly Asn Leu Arg Pro 740 745 750 Val Leu Ala Glu Asp Ala Gin Arg Leu Phe Thr Ala Leu Phe Pro 755 760 765

Phe Glu Lys Asn Cys Gly Asn Asp Asn He Cys Gin Asp Asp Leu 770 775 780

Ser He Thr Phe Ser Phe Met Ser Leu Asp Cys Leu Val Val Gly 785 790 795

Gly Pro Arg Glu Ser Asn Val Thr Val Thr Val Arg Asn Asp Gly 800 805 810

Glu Asp Ser Tyr Arg Thr Gin Val Thr Phe Phe Phe Pro Leu Asp 815 820 825

Leu Ser Tyr Arg Lys Val Ser Thr Leu Gin Asn Gin Arg Ser Gin 830 835 840

Arg Ser Trp Arg Leu Ala Cys Glu Ser Ala Ser Ser Thr Glu Val 845 850 855

Ser Gly Ala Leu Lys Ser Thr Ser Cys Ser He Asn His Pro He 860 865 870

Phe Pro Glu Asn Ser Glu Val Thr Phe Asn He Thr Phe Asp Val 875 880 885

Asp Ser Lys Ala Ser Leu Gly Asn Lys Leu Leu Leu Lys Ala Asn 890 895 900

Val Thr Ser Glu Asn Asn Met Pro Arg Thr Asn Lys Thr Glu Phe 905 910 915

Gin Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Met Val Val Thr 920 925 930

Ser His Gly Val Ser Thr Lys Tyr Leu Asn Phe Thr Ala Ser Glu 935 940 945

Asn Thr Ser Arg Val Met Gin His Gin Tyr Gin Val Ser Asn Leu 950 955 960

Gly Gin Arg Ser Pro Pro He Ser Leu Val Phe Leu Val Pro Val 965 970 975

Arg Leu Asn Gin Thr Val He Trp Asp Arg Pro Gin Val Thr Phe 980 985 990

Ser Glu Asn Leu Ser Ser Thr Cys His Thr Lys Glu Arg Leu Pro 995 1000 1005

Ser His Ser Asp Phe Leu Ala Glu Leu Arg Lys Ala Pro Val Val 1010 1015 1020

Asn Cys Ser He Ala Val Cys Gin Arg He Gin Cys Asp He Pro 1025 1030 1035

Phe Phe Gly He Gin Glu Glu Phe Asn Ala Thr Leu Lys Gly Asn 1040 1045 1050

Leu Ser Phe Asp Trp Tyr He Lys Thr Ser His Asn His Leu Leu 1055 1060 1065 He Val Ser Thr Ala Glu He Leu Phe Asn Asp Ser Val Phe Thr 1070 1075 1080

Leu Leu Pro Gly Gin Gly Ala Phe Val Arg Ser Gin Thr Glu Thr 1085 1090 1095

Lys Val Glu Pro Phe Glu Val Pro Asn Pro Leu Pro Leu He Val 1100 1105 1110

Gly Ser Ser Val Gly Gly Leu Leu Leu Leu Ala Leu He Thr Ala 1115 1120 1125

Ala Leu Tyr Lys Leu Gly Phe Phe Lys Arg Gin Tyr Lys Asp Met 1130 1135 1140

Met Ser Glu Gly Gly Pro Pro Gly Ala Glu Pro Gin 1145 1150

(2) INFORMATION FOR SEQ ID NO: 44 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1163

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 44

Met Thr Arg Thr Arg Ala Ala Leu Leu Leu Phe Thr Ala Leu Ala 5 10 15

Thr Ser Leu Gly Phe Asn Leu Asp Thr Glu Glu Leu Thr Ala Phe 20 25 30

Arg Val Asp Ser Ala Gly Phe Gly Asp Ser Val Val Gin Tyr Ala 35 40 50

Asn Ser Trp Val Val Val Gly Ala Pro Gin Lys He Thr Ala Ala 55 60 65

Asn Gin Thr Gly Gly Leu Tyr Gin Cys Gly Tyr Ser Thr Gly Ala 70 75 80

Cys Glu Pro He Gly Leu Gin Val Pro Pro Glu Ala Val Asn Met 85 90 95

Ser Leu Gly Leu Ser Leu Ala Ser Thr Thr Ser Pro Ser Gin Leu 100 105 115

Leu Ala Cys Gly Pro Thr Val His His Glu Cys Gly Arg Asn Met 120 125 130

Tyr Leu Thr Gly Leu Cys Phe Leu Leu Gly Pro Thr Gin Leu Thr 135 140 145

Gin Arg Leu Pro Val Ser Arg Gin Glu Cys Pro Arg Gin Glu Gin 150 155 160

Asp He Val Phe Leu He Asp Gly Ser Gly Ser He Ser Ser Arg 165 170 175 Asn Phe Ala Thr Met Met Asn Phe Val Arg Ala Val He Ser Gin 180 185 190

Phe Gin Arg Pro Ser Thr Gin Phe Ser Leu Met Gin Phe Ser Asn 195 200 205

Lys Phe Gin Thr His Phe Thr Phe Glu Glu Phe Arg Arg Thr Ser 215 220 225

Asn Pro Leu Ser Leu Leu Ala Ser Val His Gin Leu Gin Gly Phe 230 235 240

Thr Tyr Thr Ala Thr Ala He Gin Asn Val Val His Arg Leu Phe 245 250 255

His Ala Ser Tyr Gly Ala Arg Arg Asp Ala Thr Lys He Leu He 260 265 270

Val He Thr Asp Gly Lys Lys Glu Gly Asp Ser Leu Asp Tyr Lys 275 280 285

Asp Val He Pro Met Ala Asp Ala Ala Gly He He Arg Tyr Ala 290 295 300

He Gly Val Gly Leu Ala Phe Gin Asn Arg Asn Ser Trp Lys Glu 305 310 315

Leu Asn Asp He Ala Ser Lys Pro Ser Gin Glu His He Phe Lys 320 325 330

Val Glu Asp Phe Asp Ala Leu Lys Asp He Gin Asn Gin Leu Lys 335 340 345

Glu Lys He Phe Ala He Glu Gly Thr Glu Thr Thr Ser Ser Ser 350 355 360

Ser Phe Glu Leu Glu Met Ala Gin Glu Gly Phe Ser Ala Val Phe 365 370 375

Thr Pro Asp Gly Pro Val Leu Gly Ala Val Gly Ser Phe Thr Trp 380 385 390

Ser Gly Gly Ala Phe Leu Tyr Pro Pro Asn Met Ser Pro Thr Phe 395 400 405

He Asn Met Ser Gin Glu Asn Val Asp Met Arg Asp Ser Tyr Leu 415 420 425

Gly Tyr Ser Thr Glu Leu Ala Leu Trp Lys Gly Val Gin Ser Leu 430 435 440

Val Leu Gly Ala Pro Arg Tyr Gin His Thr Gly Lys Ala Val He 445 450 455

Phe Thr Gin Val Ser Arg Gin Trp Arg Met Lys Ala Glu Val Thr 460 465 470

Gly Thr Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys Ser Val 475 480 485

Asp Val Asp Thr Asp Gly Ser Thr Asp Leu Val Leu He Gly Ala 490 495 500 - 75 -

Pro His Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys 505 510 515

Pro Leu Pro Arg Gly Trp Arg Arg Trp Trp Cys Asp Ala Val Leu 520 525 530

Tyr Gly Glu Gin Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu 535 540 545

Thr Val Leu Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Val 550 555 560

He Gly Ala Pro Gly Glu Glu Glu Asn Arg Gly Ala Val Tyr Leu 565 570 575

Phe His Gly Val Leu Gly Pro Ser He Ser Pro Ser His Ser Gin 580 585 590

Arg He Ala Gly Ser Gin Leu Ser Ser Arg Leu Gin Tyr Phe Gly 595 600 605

Gin Ala Leu Ser Gly Gly Gin Asp Leu Thr Gin Asp Gly Leu Val 610 615 620

Asp Leu Ala Val Gly Ala Arg Gly Gin Val Leu Leu Leu Arg Thr 625 630 635

Arg Pro Val Leu Trp Val Gly Val Ser Met Gin Phe He Pro Ala 640 645 650

Glu He Pro Arg Ser Ala Phe Glu Cys Arg Glu Gin Val Val Ser 655 670 675

Glu Gin Thr Leu Val Gin Ser Asn He Cys Leu Tyr He Asp Lys 680 685 690

Arg Ser Lys Asn Leu Leu Gly Ser Arg Asp Leu Gin Ser Ser Val 695 670 675

Thr Leu Asp Leu Ala Leu Asp Pro Gly Arg Leu Ser Pro Arg Ala 680 685 690

Thr Phe Gin Glu Thr Lys Asn Arg Ser Leu Ser Arg Val Arg Val 695 700 705

Leu Gly Leu Lys Ala His Cys Glu Asn Phe Asn Leu Leu Leu Pro 710 715 720

Ser Cys Val Glu Asp Ser Val Thr Pro He Thr Leu Arg Leu Asn 725 730 735

Phe Thr Leu Val Gly Lys Pro Leu Leu Ala Phe Arg Asn Leu Arg 740 745 750

Pro Met Leu Ala Ala Leu Ala Gin Arg Tyr Phe Thr Ala Ser Leu 755 760 765

Pro Phe Glu Lys Asn Cys Gly Ala Asp His He Cys Gin Asp Asn 770 775 780

Leu Gly He Ser Phe Ser Phe Pro Gly Leu Lys Ser Leu Leu Val 785 790 795 Gly Ser Asn Leu Glu Leu Asn Ala Glu Val Met Val Trp Asn Asp 800 805 810

Gly Glu Asp Ser Tyr Gly Thr Thr He Thr Phe Ser His Pro Ala 815 820 825

Gly Leu Ser Tyr Arg Tyr Val Ala Glu Gly Gin Lys Gin Gly Gin 830 835 840

Leu Arg Ser Leu His Leu Thr Cys Asp Ser Ala Pro Val Gly Ser 845 850 855

Gin Gly Thr Trp Ser Thr Ser Cys Arg He Asn His Leu He Phe 860 865 870

Arg Gly Gly Ala Gin He Thr Phe Leu Ala Thr Phe Asp Val Ser 875 880 885

Pro Lys Ala Val Leu Gly Asp Arg Leu Leu Leu Thr Ala Asn Val 890 895 900

Ser Ser Glu Asn Asn Thr Pro Arg Thr Ser Lys Thr Thr Phe Gin 905 910 915

Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Thr Val Val Ser Ser 920 925 930

His Glu Gin Phe Thr Lys Tyr Leu Asn Phe Ser Glu Ser Glu Glu 935 940 945

Lys Glu Ser His Val Ala Met His Arg Tyr Gin Val Asn Asn Leu 950 955 960

Gly Gin Arg Asp Leu Pro Val Ser He Asn Phe Trp Val Pro Val 965 970 975

Glu Leu Asn Gin Glu Ala Val Trp Met Asp Val Glu Val Ser His 980 985 990

Pro Gin Asn Pro Ser Leu Arg Cys Ser Ser Glu Lys He Ala Pro 995 1000 1005

Pro Ala Ser Asp Phe Leu Ala His He Gin Lys Asn Pro Val Leu 1010 1015 1020

Asp Cys Ser He Ala Gly Cys Leu Arg Phe Arg Cys Asp Val Pro 1025 1030 1035

Ser Phe Ser Val Gin Glu Glu Leu Asp Phe Thr Leu Lys Gly Asn 1040 1045 1050

Leu Ser Phe Gly Trp Val Arg Gin He Leu Gin Lys Lys Val Ser 1055 1060 1065

Val Val Ser Val Ala Glu He Thr Phe Asp Thr Ser Val Tyr Ser 1070 1075 1080

Gin Leu Pro Gly Gin Glu Ala Phe Met Arg Ala Gin Thr Thr Thr 1085 1090 1095

Val Leu Glu Lys Tyr Lys Val His Asn Pro Thr Pro Leu He Val 1100 1105 1110 Gly Ser Ser He Gly Gly Leu Leu Leu Leu Ala Leu He Thr Ala 1115 1120 1125

Val Leu Tyr Lys Val Gly Phe Phe Lys Arg Gin Tyr Lys Glu Met 1130 1135 1140

Met Glu Glu Ala Asn Gly Gin He Ala Pro Glu Asn Gly Thr Gin 1145 1150 1155

Thr Pro Ser Pro Pro Ser Glu Lys 1160

(2) INFORMATION FOR SEQ ID NO: 45 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 769

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 45

Met Leu Gly Leu Arg Pro Pro Leu Leu Ala Leu Val Gly Leu Leu 5 10 15

Ser Leu Gly Cys Val Leu Ser Gin Glu Cys Thr Lys Phe Lys Val 20 25 30

Ser Ser Cys Arg Glu Cys He Glu Ser Gly Pro Gly Cys Thr Trp 35 40 50

Cys Gin Lys Leu Asn Phe Thr Gly Pro Gly Asp Pro Asp Ser He 55 60 65

Arg Cys Asp Thr Arg Pro Gin Leu Leu Met Arg Gly Cys Ala Ala 70 75 80

Asp Asp He Met Asp Pro Thr Ser Leu Ala Glu Thr Gin Glu Asp 85 90 95

His Asn Gly Gly Gin Lys Gin Leu Ser Pro Gin Lys Val Thr Leu 100 105 115

Tyr Leu Arg Pro Gly Gin Ala Ala Ala Phe Asn Val Thr Phe Arg 120 125 130

Arg Ala Lys Gly Tyr Pro He Asp Leu Tyr Tyr Leu Met Asp Leu 135 140 145

Ser Tyr Ser Met Leu Asp Asp Leu Arg Asn Val Lys Lys Leu Gly 150 155 160

Gly Asp Leu Leu Arg Ala Leu Asn Glu He Thr Glu Ser Gly Arg 165 170 175

He Gly Phe Gly Ser Phe Val Asp Lys Thr Val Leu Pro Phe Val 180 185 190

Asn Thr His Pro Asp Lys Leu Arg Asn Pro Cys Pro Asn Lys Glu 195 200 205

Lys Glu Cys Gin Pro Pro Phe Ala Phe Arg His Val Leu Lys Leu 215 220 225

Thr Asn Asn Ser Asn Gin Phe Gin Thr Glu Val Gly Lys Gin Leu 230 235 240

He Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly Leu Asp Ala Met 245 250 255

Met Gin Val Ala Ala Cys Pro Glu Glu He Gly Trp Arg Asn Val 260 265 270

Thr Arg Leu Leu Val Phe Ala Thr Asp Asp Gly Phe His Phe Ala 275 280 285

Gly Asp Gly Lys Leu Gly Ala He Leu Thr Pro Asn Asp Gly Arg 290 295 300

Cys His Leu Glu Asp Asn Leu Tyr Lys Arg Ser Asn Glu Phe Asp 305 310 315

Tyr Pro Ser Val Gly Gin Leu Ala His Lys Leu Ala Glu Asn Asn 320 325 330

He Gin Pro He Phe Ala Val Thr Ser Arg Met Val Lys Thr Tyr 335 340 345

Glu Lys Leu Thr Glu He He Pro Lys Ser Ala Val Gly Glu Leu 350 355 360

Ser Glu Asp Ser Ser Asn Val Val His Leu He Lys Asn Ala Tyr 365 370 375

Asn Lys Leu Ser Ser Arg Val Phe Leu Asp His Asn Ala Leu Pro 380 385 390

Asp Thr Leu Lys Val Thr Tyr Asp Ser Phe Cys Ser Asn Gly Val 395 400 405

Thr His Arg Asn Gin Pro Arg Gly Asp Cys Asp Gly Val Gin He 415 420 425

Asn Val Pro He Thr Phe Gin Val Lys Val Thr Ala Thr Glu Cys 430 435 440

He Gin Glu Gin Ser Phe Val He Arg Ala Leu Gly Phe Thr Asp 445 450 455

He Val Thr Val Gin Val Leu Pro Gin Cys Glu Cys Arg Cys Arg 460 465 470

Asp Gin Ser Arg Asp Arg Ser Leu Cys His Gly Lys Gly Phe Leu 475 480 485

Glu Cys Gly He Cys Arg Cys Asp Thr Gly Tyr He Gly Lys Asn 490 495 500

Cys Glu Cys Gin Thr Gin Gly Arg Ser Ser Gin Glu Leu Glu Gly 505 510 515

Ser Cys Arg Lys Asp Asn Asn Ser He He Cys Ser Gly Leu Gly 520 525 530

Asp Cys Val Cys Gly Gin Cys Leu Cys His Thr Ser Asp Val Pro 535 540 545

Gly Lys Leu He Tyr Gly Gin Tyr Cys Glu Cys Asp Thr He Asn 550 555 560

Cys Glu Arg Tyr Asn Gly Gin Val Cys Gly Gly Pro Gly Arg Gly 565 570 575

Leu Cys Phe Cys Gly Lys Cys Arg Cys His Pro Gly Phe Glu Gly 580 585 590

Ser Ala Cys Gin Cys Glu Arg Thr Thr Glu Gly Cys Leu Asn Pro 595 600 605

Arg Arg Val Glu Cys Ser Gly Arg Gly Arg Cys Arg Cys Asn Val 610 615 620

Cys Glu Cys His Ser Gly Tyr Gin Leu Pro Leu Cys Gin Glu Cys 625 630 635

Pro Gly Cys Pro Ser Pro Cys Gly Lys Tyr He Ser Cys Ala Glu 640 645 650

Cys Leu Lys Phe Glu Lys Gly Pro Phe Gly Lys Asn Cys Ser Ala 655 670 675

Ala Cys Pro Gly Leu Gin Leu Ser Asn Asn Pro Val Lys Gly Arg 680 685 690

Thr Cys Lys Glu Arg Asp Ser Glu Gly Cys Trp Val Ala Tyr Thr 695 670 675

Leu Glu Gin Gin Asp Gly Met Asp Arg Tyr Leu He Tyr Val Asp 680 685 690

Glu Ser Arg Glu Cys Val Ala Gly Pro Asn He Ala Ala He Val 695 700 705

Gly Gly Thr Val Ala Gly He Val Leu He Gly He Leu Leu Leu 710 715 720

Val He Trp Lys Ala Leu He His Leu Ser Asp Leu Arg Glu Tyr 725 730 735

Arg Arg Phe Glu Lys Glu Lys Leu Lys Ser Gin Trp Asn Asn Asp 740 745 750

Asn Pro Leu Phe Lys Ser Ala Thr Thr Thr Val Met Asn Pro Lys 755 760 765

Phe Ala Glu Ser

(2) INFORMATION FOR SEQ ID NO: 46 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid (C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 46 sp Val Asp Ser Asn Gly Ser Thr Asp

5

(2) INFORMATION FOR SEQ ID NO: 47 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 47 sp Val Asn Gly Asp Lys Leu Thr Asp 5

(2) INFORMATION FOR SEQ ID NO: 48 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 48 sp Leu Thr Met Asp Gly Leu Val Asp 5

(2) INFORMATION FOR SEQ ID NO: 49 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 49 sp Ser Asp Met Asn Asp Ala Tyr Leu

5

(2) INFORMATION FOR SEQ ID NO: 50 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 (B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 50

Asn Ala Phe Lys He Leu Val Val He Thr Asp Gly Glu Lys Phe 5 10 15

Gly Asp Pro Leu Gly Tyr Glu Asp Val He Pro Glu Ala Asp Arg 20 25 30

Glu Gly Val

(2) INFORMATION FOR SEQ ID NO: 51 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 51

Asp Gly Glu Lys Phe 5

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 253 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

Gly Ala Glu Ala Asn Phe Met Leu Lys Val His Pro Leu Lys Lys Tyr 1 5 10 15

Pro Val Asp Leu Tyr Tyr Leu Val Asp Val Ser Ala Ser Met His Asn 20 25 30

Asn He Glu Lys Leu Asn Ser Val Gly Asn Asp Leu Ser Arg Lys Met 35 40 45

Ala Phe Phe Ser Arg Asp Phe Arg Leu Gly Phe Gly Ser Tyr Val Asp 50 55 60

Lys Thr Val Ser Pro Tyr He Ser He His Pro Glu Arg He His Asn 65 70 75 80 Gin Cys Ser Asp Tyr Asn Leu Asp Cys Met Pro Pro His Gly Tyr He 85 90 95

His Val Leu Ser Leu Thr Glu Asn He Thr Glu Phe Glu Lys Ala Val 100 105 110

His Arg Gin Lys He Ser Gly Asn He Asp Thr Pro Glu Gly Gly Phe 115 120 125

Asp Ala Met Leu Gin Ala Ala Val Cys Glu Ser His He Gly Trp Arg 130 135 140

Lys Glu Ala Lys Arg Leu Leu Leu Val Met Thr Asp Gin Thr Ser His 145 150 155 160

Leu Ala Leu Asp Ser Lys Leu Ala Gly He Val Val Pro Asn Asp Gly 165 170 175

Asn Cys His Leu Lys Asn Asn Val Tyr Val Lys Ser Thr Thr Met Glu 180 185 190

His Pro Ser Leu Gly Gin Leu Ser Glu Lys Leu He Asp Asn Asn He 195 200 205

Asn Val He Phe Ala Val Gin Gly Lys Gin Phe His Trp Tyr Lys Asp 210 215 220

Leu Leu Pro Leu Leu Pro Gly Thr He Ala Gly Glu He Glu Ser Lys 225 230 235 240

Ala Ala Asn Leu Asn Asn Leu Val Val Glu Ala Tyr Gin 245 250

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 253 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

Gly Glu Pro Gin Gin Leu Gin Val Arg Phe Leu Arg Ala Glu Gly Tyr 1 5 10 15

Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser Ser Ser Met Lys Asp 20 25 30

Asp Leu Glu Arg Val Arg Gin Leu Gly His Ala Leu Leu Val Arg Leu 35 40 45

Gin Glu Val Thr His Ser Val Arg He Gly Phe Gly Ser Phe Val Asp 50 55 60

Lys Thr Val Leu Pro Phe Val Ser Thr Val Pro Ser Lys Leu Arg His 65 70 75 80 Pro Cys Pro Thr Arg Leu Glu Arg Cys Gin Ser Pro Phe Ser Phe His 85 90 95

His Val Leu Ser Leu Thr Gly Asp Ala Gin Ala Phe Glu Arg Glu Val 100 105 110

Gly Arg Gin Ser Val Ser Gly Asn Leu Asp Ser Pro Glu Gly Gly Phe 115 120 125

Asp Ala He Leu Gin Ala Ala Leu Cys Gin Glu Gin He Gly Trp Arg 130 135 140

Asn Val Ser Arg Leu Leu Val Phe Thr Ser Asp Asp Thr Phe His Thr 145 150 155 160

Ala Gly Asp Gly Lys Leu Gly Gly He Phe Met Pro Ser Asp Gly Ser

165 170 175

Cys His Leu Asp Ser Asn Gly Leu Tyr Ser Arg Ser Thr Glu Phe Asp 180 185 190

Tyr Pro Ser Val Gly Gin Val Ala Gin Ala Leu Ser Ala Ala Asn He 195 200 205

Gin Pro He Phe Ala Val Thr Ser Ala Ala Leu Pro Val Tyr Gin Glu 210 215 220

Leu Ser Lys Leu He Pro Lys Ser Ala Val Gly Glu Leu Ser Glu Asp 225 230 235 240

Ser Ser Asn Val Val Gin Leu He Met Asp Ala Tyr Asn 245 250

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 255 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

Gly Gly Ala Gin Thr Leu Gin Val His Val Arg Gin Thr Glu Asp Tyr 1 5 10 15

Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser Ala Ser Met Asp Asp 20 25 30

Asp Leu Asn Thr He Lys Glu Leu Gly Ser Gly Leu Ser Lys Glu Met 35 40 45

Ser Lys Leu Thr Ser Asn Phe Arg Leu Gly Phe Gly Ser Phe Val Glu 50 55 60

Lys Pro Val Ser Pro Phe Val Lys Thr Thr Pro Glu Glu He Ala Asn 65 70 75 80 - 84 -

Pro Cys Ser Ser He Pro Tyr Phe Cys Leu Pro Thr Phe Gly Phe Lys 85 90 95

His He Leu Pro Leu Thr Asn Asp Ala Glu Arg Phe Asn Glu He Val 100 105 110

Lys Asn Gin Lys He Ser Ala Asn He Asp Thr Pro Glu Gly Gly Phe 115 120 125

Asp Ala He Met Gin Ala Ala Val Cys Lys Glu Lys He Gly Trp Trp 130 135 140

Arg Asn Asp Ser Leu His Leu Leu Val Phe Val Ser Asp Ala Asp Ser 145 150 155 160

His Phe Gly Met Asp Ser Lys Leu Ala Gly He Val He Pro Asn Asp 165 170 175

Gly Leu Cys His Leu Asp Ser Lys Asn Glu Tyr Ser Met Ser Thr Val 180 185 190

Leu Glu Tyr Pro Thr He Gly Gly Leu He Asp Lys Leu Val Gin Asn 195 200 205

Asn Val Leu Leu He Phe Ala Val Thr Gin Glu Gin Val His Leu Tyr 210 215 220

Glu Asn Tyr Ala Lys Leu He Pro Gly Ala Thr Val Gly Leu Leu Gin 225 230 235 240

Lys Asp Ser Gly Asn He Leu Gin Leu He He Ser Ala Tyr Glu 245 250 255

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 256 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

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

Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser Leu Ser Met Lys Asp 20 25 30

Asp Leu Asp Asn He Arg Ser Leu Gly Thr Lys Leu Ala Glu Glu Met 35 40 45

Arg Lys Leu Thr Ser Asn Phe Arg Leu Gly Phe Gly Ser Phe Val Asp 50 55 60

Lys Asp He Ser Pro Phe Ser Tyr Thr Ala Pro Arg Tyr Gin Thr Asn 65 70 75 80 - 85 -

Pro Cys He Gly Tyr Lys Leu Phe Pro Asn Cys Val Pro Ser Phe Gly 85 90 95

Phe Arg His Leu Leu Pro Leu Thr Asp Arg Val Asp Ser Phe Asn Glu 100 105 110

Glu Val Arg Lys Gin Arg Val Ser Arg Asn Arg Asp Ala Pro Glu Gly 115 120 125

Gly Phe Asp Ala Val Leu Gin Ala Ala Val Cys Lys Glu Lys He Gly 130 135 140

Trp Arg Lys Asp Ala Leu His Leu Leu Val Phe Thr Thr Asp Asp Val 145 150 155 160

Pro His He Ala Leu Asp Gly Lys Leu Gly Gly Leu Val Gin Pro His 165 170 175

Asp Gly Gin Cys His Leu Asn Glu Ala Asn Glu Tyr Thr Ala Ser Asn 180 185 190

Gin Met Asp Tyr Pro Ser Leu Ala Leu Leu Gly Glu Lys Leu Ala Glu 195 200 205

Asn Asn He Asn Leu He Phe Ala Val Thr Lys Asn His Tyr Met Leu 210 215 220

Tyr Lys Asn Phe Thr Ala Leu He Pro Gly Thr Thr Val Glu He Leu 225 230 235 240

Asp Gly Asp Ser Lys Asn He He Gin Leu He He Asn Ala Tyr Asn 245 250 255

(2) INFORMATION FOR SEQ ID NO:56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 252 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

Gly Glu Glu Arg His Phe Glu Leu Glu Val Phe Glu Pro Leu Glu Ser 1 5 10 15

Pro Val Asp Leu Tyr He Leu Met Asp Phe Ser Asn Ser Met Ser Asp 20 25 30

Asp Leu Asp Asn Leu Lys Lys Met Gly Gin Asn Leu Ala Arg Val Leu 35 40 45

Ser Gin Leu Thr Ser Asp Tyr Thr He Gly Phe Gly Lys Phe Val Asp 50 55 60

Lys Val Ser Val Pro Gin Thr Asp Met Arg Pro Glu Lys Leu Lys Glu 65 70 75 80 Pro Trp Pro Asn Ser Asp Pro Pro Phe Ser Phe Lys Asn Val He Ser 85 90 95

Leu Thr Glu Asp Val Asp Glu Phe Arg Asn Lys Leu Gin Gly Glu Arg 100 105 110

He Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly Phe Asp Ala He Leu 115 120 125

Gin Thr Ala Val Cys Thr Arg Asp He Gly Trp Arg Pro Asp Ser Thr 130 135 140

His Leu Leu Val Phe Ser Thr Glu Ser Ala Phe His Tyr Glu Ala Asp 145 150 155 160

Gly Ala Asn Val Leu Ala Gly He Met Ser Arg Asn Asp Glu Arg Cys 165 170 175

His Leu Asp Thr Thr Gly Thr Tyr Thr Gin Tyr Arg Thr Gin Asp Tyr 180 185 190

Pro Ser Val Pro Thr Leu Val Arg Leu Leu Ala Lys His Asn He He 195 200 205

Pro He Phe Ala Val Thr Asn Tyr Ser Tyr Ser Tyr Tyr Glu Lys Leu 210 215 220

His Thr Tyr Phe Pro Val Ser Ser Leu Gly Val Leu Gin Glu Asp Ser 225 230 235 240

Ser Asn He Val Glu Leu Leu Glu Glu Ala Phe Asn 245 250

(2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 255 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

Asp Asp Ser Lys Asn Phe Ser He Gin Val Arg Gin Val Glu Asp Tyr 1 5 10 15

Pro Val Asp He Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Lys Asp 20 25 30

Asp Leu Trp Ser He Gin Asn Leu Gly Thr Lys Leu Ala Thr Gin Met 35 40 45

Arg Lys Leu Thr Ser Asn Leu Arg He Gly Phe Gly Ala Phe Val Asp 50 55 60

Lys Pro Val Ser Pro Tyr Met Tyr He Ser Pro Pro Glu Ala Leu Glu 65 70 75 80 Asn Pro Cys Tyr Asp Met Lys Thr Thr Cys Leu Pro Met Phe Gly Tyr 85 90 95

Lys His Val Leu Thr Leu Thr Asp Gin Val Thr Arg Phe Asn Glu Glu 100 105 110

Val Lys Lys Gin Ser Val Ser Arg Asn Arg Asp Ala Pro Glu Gly Gly 115 120 125

Phe Asp Ala He Met Gin Ala Thr Val Cys Asp Glu Lys He Gly Trp 130 135 140

Arg Asn Asp Ala Ser His Leu Leu Val Phe Thr Thr Asp Ala Lys Thr 145 150 155 160

His He Ala Leu Asp Gly Arg Leu Ala Gly He Val Gin Pro Asn Asp 165 170 175

Gly Gin Cys His Val Gly Ser Asp Asn His Tyr Ser Ala Ser Thr Thr 180 185 190

Met Asp Tyr Pro Ser Leu Gly Leu Met Thr Glu Lys Leu Ser Gin Lys 195 200 205

Asn He Asn Leu He Phe Ala Val Thr Glu Asn Val Val Asn Leu Tyr 210 215 220

Gin Asn Tyr Ser Glu Leu He Pro Gly Thr Thr Val Gly Val Leu Ser 225 230 235 240

Met Asp Ser Ser Asn Val Leu Gin Leu He Val Asp Ala Tyr Gly 245 250 255

(2) INFORMATION FOR SEQ ID NO:58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 252 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

Gly Gin Ala Ala Ala Phe Asn Val Thr Phe Arg Arg Ala Lys Gly Tyr

1 5 10 15

Pro He Asp Leu Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Leu Asp 20 25 30

Asp Leu Arg Asn Val Lys Lys Leu Gly Gly Asp Leu Leu Arg Ala Leu 35 40 45

Asn Glu He Thr Glu Ser Gly Arg He Gly Phe Gly Ser Phe Val Asp 50 55 60

Lys Thr Val Leu Pro Phe Val Asn Thr His Pro Asp Lys Leu Arg Asn 65 70 75 80 Pro Cys Pro Asn Lys Glu Lys Glu Cys Gin Pro Pro Phe Ala Phe Arg 85 90 95

His Val Leu Lys Leu Thr Asn Asn Ser Asn Gin Phe Gin Thr Glu Val 100 105 110

Gly Lys Gin Leu He Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly Leu 115 120 125

Asp Ala Met Met Gin Val Ala Ala Cys Pro Glu Glu He Gly Trp Arg 130 135 140

Asn Val Thr Arg Leu Leu Val Phe Ala Thr Asp Asp Gly Phe His Phe 145 150 155 160

Ala Gly Asp Gly Lys Leu Gly Ala He Leu Thr Pro Asn Asp Gly Arg 165 170 175

Cys His Leu Glu Asp Asn Leu Tyr Lys Arg Ser Asn Glu Phe Asp Tyr 180 185 190

Pro Ser Val Gly Gin Leu Ala His Lys Leu Ala Glu Asn Asn He Gin 195 200 205

Pro He Phe Ala Val Thr Ser Arg Met Val Lys Thr Tyr Glu Lys Leu 210 215 220

Thr Glu He He Pro Lys Ser Ala Val Gly Glu Leu Ser Glu Asp Ser 225 230 235 240

Ser Asn Val Val His Leu He Lys Asn Ala Tyr Asn 245 250

(2) INFORMATION FOR SEQ ID NO:59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 251 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

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

Pro He Asp Leu Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Lys Asp 20 25 30

Asp Leu Glu Asn Val Lys Ser Leu Gly Thr Asp Leu Met Asn Glu Met 35 40 45

Arg Arg He Thr Ser Asp Phe Arg He Gly Phe Gly Ser Phe Val Glu 50 55 60

Lys Thr Val Met Pro Tyr He Ser Thr Thr Pro Ala Lys Leu Arg Asn 65 70 75 80 Pro Cys Thr Ser Glu Gin Asn Cys Thr Thr Pro Phe Ser Tyr Lys Asn 85 90 95

Val Leu Ser Leu Thr Asn Lys Gly Glu Val Phe Asn Glu Leu Val Gly 100 105 110

Lys Gin Arg He Ser Gly Asn Leu Asp Ser Pro Glu Gly Gly Phe Asp

115 120 125

Ala He Met Gin Val Ala Val Cys Gly Ser Leu He Gly Trp Arg Asn 130 135 140

Val Thr Arg Leu Leu Val Phe Ser Thr Asp Ala Gly Phe His Phe Ala 145 150 155 160

Gly Asp Gly Lys Leu Gly Gly He Val Leu Pro Asn Asp Gly Gin Cys 165 170 175

His Leu Glu Asn Asn Met Tyr Thr Met Ser His Tyr Tyr Asp Tyr Pro 180 185 190

Ser He Ala His Leu Val Gin Lys Leu Ser Glu Asn Asn He Gin Thr 195 200 205

He Phe Ala Val Thr Glu Glu Phe Gin Pro Val Tyr Lys Glu Leu Lys 210 215 220

Asn Leu He Pro Lys Ser Ala Val Gly Thr Leu Ser Ala Asn Ser Ser 225 230 235 240

Asn Val He Gin Leu He He Asp Ala Tyr Asn 245 250

(2) INFORMATION FOR SEQ ID NO:60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 187 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:

Cys Pro Arg Gin Glu Gin Asp He Val Phe Leu He Asp Gly Ser Gly 1 5 10 15

Ser He Ser Ser Arg Asn Phe Ala Thr Met Met Asn Phe Val Arg Ala 20 25 30

Val He Ser Gin Phe Gin Arg Pro Ser Thr Gin Phe Ser Leu Met Gin 35 40 45

Phe Ser Asn Lys Phe Gin Thr His Phe Thr Phe Glu Glu Phe Arg Arg 50 55 60

Thr Ser Asn Pro Leu Ser Leu Leu Ala Ser Val His Gin Leu Gin Gly 65 70 75 80 Phe Thr Tyr Thr Ala Thr Ala He Gin Asn Val Val His Arg Leu Phe 85 90 95

His Ala Ser Tyr Gly Ala Arg Arg Asp Ala Thr Lys He Leu He Val 100 105 110

He Thr Asp Gly Lys Lys Glu Gly Asp Ser Leu Asp Tyr Lys Asp Val 115 120 125

He Pro Met Ala Asp Ala Ala Gly He He Arg Tyr Ala He Gly Val 130 135 140

Gly Leu Ala Phe Gin Asn Arg Asn Ser Trp Lys Glu Leu Asn Asp He 145 150 155 160

Ala Ser Lys Pro Ser Gin Glu His He Phe Lys Val Glu Asp Phe Asp 165 170 175

Ala Leu Lys Asp He Gin Asn Gin Leu Lys Glu 180 185

(2) INFORMATION FOR SEQ ID NO:61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 181 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:

Cys He Lys Gly Asn Val Asp Leu Val Phe Leu Phe Asp Gly Ser Met 1 5 10 15

Ser Leu Gin Pro Asp Glu Phe Gin Lys He Leu Asp Phe Met Lys Asp 20 25 30

Val Met Lys Lys Leu Ser Asn Thr Ser Tyr Gin Phe Ala Ala Val Gin 35 40 45

Phe Ser Thr Ser Tyr Lys Thr Glu Phe Asp Phe Ser Asp Tyr Val Lys 50 55 60

Trp Lys Asp Pro Asp Ala Leu Leu Lys His Val Lys His Met Leu Leu 65 70 75 80

Leu Thr Asn Thr Phe Gly Ala He Asn Tyr Val Ala Thr Glu Val Phe 85 90 95

Arg Glu Glu Leu Gly Ala Arg Pro Asp Ala Thr Lys Val Leu He He 100 105 110

He Thr Asp Gly Glu Ala Thr Asp Ser Gly Asn He Asp Ala Ala Lys 115 120 125

Asp He He Arg Tyr He He Gly He Gly Lys His Phe Gin Thr Lys 130 135 140 Glu Ser Gin Glu Thr Leu His Lys Phe Ala Ser Lys Pro Ala Ser Glu 145 150 155 160

Phe Val Lys He Leu Asp Thr Phe Phe Glu Lys Leu Lys Asp Leu Phe 165 170 175

He Glu Arg Gin Lys 180

(2) INFORMATION FOR SEQ ID NO:62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE : protein

( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 62 :

Cys Pro Gin Glu Asp Ser Asp He Ala Phe Leu He Asp Gly Ser Gly 1 5 10 15

Ser He He Pro 20

(2) INFORMATION FOR SEQ ID NO:63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:

He He Pro His Asp Phe Arg Arg Met Lys Glu Phe Val Ser Thr Val 1 5 10 15

Met Glu Gin Leu 20

(2) INFORMATION FOR SEQ ID NO:64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

Glu Gin Leu Lys Lys Ser Lys Thr Leu Phe Ser Leu Met Gin Tyr Ser 1 5 10 15

Glu Glu Phe Arg 20

(2) INFORMATION FOR SEQ ID NO: 65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:

Glu Phe Arg He His Phe Thr Phe Lys Glu Phe Gin Asn Asn Pro Asn 1 5 10 15

Pro Arg Ser Leu 20

(2) INFORMATION FOR SEQ ID NO:66:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:

Arg Ser Leu Val Lys Pro He Thr Gin Leu Leu Gly Arg Thr His Thr 1 5 10 15

Ala Thr Gly He 20

(2) INFORMATION FOR SEQ ID NO:67:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 67 :

Thr Gly He Arg Lys Val Val Arg Glu Leu Phe Asn He Thr Asn Gly 1 5 10 15

Ala Arg Lys Asn

20

(2) INFORMATION FOR SEQ ID NO:68:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:

Lys Val Val Arg Glu Leu Ser Asn He Thr Asn Gly Ala Arg Lys Asn 1 5 10 15

Ala Ser Lys He Leu Val Val He Thr Asp Gly Glu Lys 20 25

(2) INFORMATION FOR SEQ ID NO:69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

Asn Ala Phe Lys He Leu Val Val He Thr Asp Gly Glu Lys Phe Gly 1 5 10 15

Asp Pro Leu Gly Tyr Glu Asp Val 20

(2) INFORMATION FOR SEQ ID NO:70:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein - 94 - ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 70 :

Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly Tyr Glu Asp Val He Pro 1 5 10 15

Glu Ala Asp Arg 20

(2) INFORMATION FOR SEQ ID NO:71:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE : protein

( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 71 :

Asp Arg Glu Gly Val He Arg Tyr Val He Gly Val Gly Asp Ala Phe 1 5 10 15

Arg

(2) INFORMATION FOR SEQ ID NO:72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:

Phe Arg Ser Glu Lys Ser Arg Gin Glu Leu Asn Thr He Ala Ser Lys

1 5 10 15

Pro Pro Arg Asp His Val 20

(2) INFORMATION FOR SEQ ID NO:73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 73 :

His Val Phe Gin Val Asn Asn Phe Glu Ala Leu Lys Thr He Gin Asn 1 5 10 15

Gin Leu Arg Glu 20

(2) INFORMATION FOR SEQ ID NO:74:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:

Asn Ala Phe Lys He Leu Val Val He Thr Asp Gly Glu Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:

Asn Ala Phe Lys He Leu Val

1 5

(2) INFORMATION FOR SEQ ID NO: 76:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:

Val He Thr Asp Gly Glu Lys 1 5 (2) INFORMATION FOR SEQ ID NO:77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:

Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly 1 5 10

(2) INFORMATION FOR SEQ ID NO:78:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:

Asp Gly Glu Lys Phe 1 5

Claims

Claims

1. A in vitro method of screening candidate compounds for the ability to inhibit the binding of a selected integrin to a selected ligand which naturally binds to said selected integrin, said method comprising: a) measuring the binding of an A-domain peptide derived from said selected integrin to said selected ligand in the presence of said candidate compound; b) measuring the binding of said A-domain peptide derived from said selected integrin to said selected ligand in the absence of said candidate compound; c) determining whether said binding is decreased in the presence of said candidate compound; d) identifying inhibiting compounds as those which decrease said binding.

2. The method of claim 1 wherein said selected integrin is a β2 integrin.

3. The method of claim 2 wherein said β2 integrin is selected from the group comprising

CDlla/CD18, CDllb/CD18, and CDllc/CDlδ.

4. The method of claim 3 wherein said β2 integrin is CDllb/CD18.

5. The method of claim 3 wherein said ,52 integrin is CDlla/CD18.

6. The method of claim 3 wherein said β2 integrin is CDllc/CD18.

7. The method of claim 2 wherein said A-domain peptide is derived from the α subunit of said selected integrin.

8. The method of claim 2 wherein in said A- domain peptide is a CDllb A-domain peptide.

9. The method of claim 2 wherein in said A- domain peptide is a CDlla A-domain peptide.

10. The method of claim 2 wherein said A-domain peptide is a CDllc A-domain peptide.

11. The method of claim 2 wherein said A-domain peptide is derived from the β subunit of said selected integrin.

12. The method of claim 1 wherein said ligand is detectably labelled.

13. A in vitro method of screening candidate compounds for the ability to bind to a selected integrin, said method comprising: a) measuring the binding of an A-domain peptide derived from said selected integrin to said candidate compound; d) identifying compounds capable of binding said selected integrin as those which bind to said A-domain peptide.