CA2152599A1 - Novel neutrophil inhibitors - Google Patents

Abstract

Compositions enriched for Neutrophil Inhibitory Factor which inhibit neutrophil activity including adhesion to vascular endothelial cells are provided. Also provided are recombinant Neutrophil Inhibitory Factors which also inhibit neutrophil activity. Such compositions may comprise a glycoprotein isolated from nematodes. These compositions and recombinant Neutrophil Inhibitory Factors are useful in the therapy of conditions which involve abnormal or undesired inflammatory responses.

Description

2 15 2 S 9 9 PCT/US93/12626 DESCRIPTION

Novel NeutroDh;l Inhibitors Cross Reference to Related ADDlications This application is a continuation-in-part of United States Serial No. 08tl51,064, filed November 10 1993, which is a continuation in-part of United States Serial No. 08/060,433 filed May 11, 1993, which is a continuation~in-part of United States Serial No.
07/996,972 filed December 24, 1992, which is a continuation-in-part of United States Serial No.
07/881,721 filed May 11, 1992, the disclosures of which are incorporated herein by reference.

Field of the Invention This invention relates to factors which interact with CDllb/CD18 ingegrin complex or the I-domain portion of CDllb/CD18 integrin complex and inhibit leukocyte activity. These factors inhibit neutrophil activity, including inhibition of neutrophil activation and adhesion of neutrophils to vascular endothelial cells.
These factors also inhibit eosinophil activity, including inhibition of eosinophil adhesion to vascular endothelial cells.

Backqround of the Invention Leukocytes are a class of cells comprised of lymphocytes, monocytes and granul~ocytes. The lymphocytes include within ~heir class, T-cells (as helper T-cells and cytotoxic or suppressor T-cell), B-cells (as circulating B-cells and plasma cells)~ third population or natural killer (NK) cells and antigen-presenting cells. Monocytes include within their class, circulating blood monocytes, Kupffer cells, intraglomerular mesangial cells, alveolar macrophages~

PCT~S93/12626 WO g4114973 "
_ 2~ 99 2 serosal macrophages, microglia, spleen sinus macrophages and lymph node sinus macrophages. Granulocytes include within their class, neutrophils, eosinophils, basophils, mast cells, (as mucosa-associated mast cells and connective tissue mast cells).
Neutrophils are an essential component of the host defense system against microbial invasion. In response to soluble inflammatory mediators released by cells at the site of injury, neutrophils emigrate into tissue from the bloodstream by crossing the blood vessel wall.
At the site~of injury, activated neutrophils kill foreign cells by phagocytosis and by the release of cytotoxic compounds, such as oxidants, proteases and cytokines. Despite their importance in fighting infection, neutrophils themselves can promote tissue damage. During an abnormal inflammatory response, neutrophils can cause significant tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue. Alternatively, neutrophils that stick to the capillary wall or clump in venules may produce tissue damage by ischemia. Such abnormal inflammatory responses have been implicated in the pathogenesis of a variety of clinical disorders including adult respiratory distress syndrome (ARDS);
ischemia-reperfusion injury following myocardial infarction, shock, stroke, and organ transplantation;
acute and chronic allograft rejection; vasculitis;
sepsis; rheumatoid arthritis; and inflammatory skin diseases (Harlan et al., 1990 Immunol. Rev. 114, 5).
Neutrophil adhesion at the site of inflammation is believed to involve at least two discrete cell-cell interactive events. Initially, vascular endothelium adjacent to inflamed tissue becomes stick~ for neutrophils; neutrophils interact with the endothelium via low affinity adhesive mechanisms in a process known as "rolling". In the second adhesive step, rolling

3 PCT~S93112626 neutrophils bind more tightly to vascular endothelial cells and migrate from the blood vessel into the tissue.
Neutrophil rolling along affected vascular segments and other initial low affinity contacts between neutrophils and the endothelium are reported to be mediated by a group of monomeric, integral membrane gly~o~o-eins termed selections. All three of the selections so far identified, that is L-selectin (LECAM-l or LAM-l) present on the surface of neutrophils, E-selectin (endothelial leukocyte adhesion molecule-l~or ELAM-l) present on endothelial cells and P-selectin (granule membrane protein-140, GMP-140, platelet activation-dep~n~nt granule-external membrane protein, PADGEM or CD62) expressed on endothelial cells, have been implicated in neu~u~hil adhesion to the vascular endothelium (Jutila et al., 1989 J. Immunol 143, 3318; Watson et al., 1991 Nature 349, 164; Mulligan et al., J. Clin. Invest. 88, 1396; Gundel et al., 1991 J. Clin. Invest. 88, 1407; Geng et al., 1990 Nature 343, 757; Patel et al., 1991 J. Cell Biol. 112, 749). The counter-receptor for E-selectin is ~e~o~Led to be the sialylated Lewis X antigen (sialyl-LewisX) that is present on cell-surface gly~Gp~oLeins (Phillips et al., 1990 Science 250, 1130; Walz et al., 1990 Science 250, 2~ 1132; Tiemeyer et al., 1991 Proc. Natl. Acad. Sci.(USA) 88, 1138; Lowe et al., 1990 Cell 63, 475). Receptors for the other selections are also thought to be carbohydrate in nature but remain to be elucidated.
The more stable secondary contacts between neutrophils and endothelial cells are reported to be mediated by a class of cell adhesion molecules known as integrins. Integrins comprise a broad range of evolutionarily conserved heterodimeric tr~smembrane glycoprotein complexes that are present on virtually all '~ cell types. Members of the leukocyte-specific CD18 (~2) family of integrins, which include CDlla/CD18 tLFA-1) WO94/14973 PCT~S9311262~
. ., ~_.

2~s~sg9 4 and CDllb/CD18 (Mac-1, Mo-1 or CR3) have been reported to mediate neuLLG~hil adhesion to the endothelium (See Larson and Springer~` 1990 Immunol Rev. 114, 181).
Endothelial cell counter-receptors for these integrins are the intercellular cell adhesion molecules ICAM-1 and ICAM-2 for CDlla/CD18 and ICAM-l for ~11~18~ respectively (Rothlein et al., 1986 J. Immunol. 137, 1270; Staunton et al., 1988 Cell 52, 925; Staunton et al., 1989 Nature 339, 61). The ICAMs are monomeric transmembrane proteins that are members of the immunoglobulin superfamily.
The CDllb/CD18 integrin is expressed on a variety of leukocytes, including monocytes, macrophages, granulocytes, large granular lymphocytes (NK cells), and immature and CDS' B cells (K;chimoto, T.K., Larson, R.S., Corbi, A.L., Dustin, M.L., Staunton, D.E., and Spriger, T.A. (1989) Adv. in Immunol. 46,149-182).
CDllb/CD18 has been implicated in a variety of leukocyte functions including adhesion of ne~L~o~hils to endothelial cells (Prieto, J., Beatty, P.G., Clark, E.A., and Patarroyo, M. (1988) Immunology 63, 631-637;
Wallis, W.J., Hickstein, D.D., Schwartz, B.R., June, C.H., Ochs, H.D., Beatty, P.G., Klebanoff, S.J., and Harlan, J.M. (1986) Blood 67, 1007-1013; Smith, C.W., Marlin, S.D., Rothlein, R., Toman, C., and Anderson, D.C. (1989) J. Clin. Invest. 83, 2008-2017) and release of hydrogen peroxide from neu~o~hils (Shappell, S.B., Toman, C., Anderson, D.C., Taylor, A.A., Entman, M.L.
and Smith, C.W. (1990) J. Immunol. 144, 2702-2711; Von Asmuth, E.J.U., Van Der Linden, C.J., Leeuwenberg, J.F.M., and Buurman, W.A. (1991) J. Immunol. 147,3869-3875). This integrin may play a roll in neutrophil and monocye phagocytosis of opsonized (ie C3bi-coated) targets (Beller, D.I., Springer, T.A., and Schreiber, R.D. (1982) J.Exp. Med. 156,1000-1009). It has also 3~ been reported that CDllb/CD18 contributes to elevated natural killer activity against C3bi-coated target cells WO94/14973 PCT~S93112626 2152~99 (Ramos, O.F., Kai, C., Yefenof, E., and Klein, E. (1988) J. Immunol. 140,1239-1243).
The activation of endothelial cells and neutrophils is believed to ~ e~L ~ent an important compQn~nt of ne~LLG~hil-mediated inflammation. Factors that induce cell activation are termed agonists. Endothelial cell agonists, which are believed to include small regulatory proteins such as tumor necrosis factor (TNF~) and interleukin-l~ (IL-l~), are released by cells at the lo site of injury. Activation of endo~h~ l cells has been report~d to result in the increased surface expression of ICAM-1 (Staunton et al., 1988 Cell 52, 925) and ELAM-1 (Bevilacqua et al., 1987 Proc. Natl.
Acad. Sci.(USA) 84, 9238). Raised levels of expression of these adhesive molecules on the surface of activated endothelial cells is believed to lead to the observed increased adhesivity of neu~L~hils for the vascular endothelium near sites of injury.
Activation of the neu~L~hil results in profound changes to its physiological state, including shape change, ability to phagocytose foreign bodies and release of cytotoxic subst~nces from intracellular granules. Moreover, activation is believed to greatly increase the affinity of adhesive contacts between neutrophils and the vascular endothelium, perhaps through a conformational change in the CDllb/CDl8 integrin complex on the neuL~o~hil surface (Vedder and Harlan, 1988 J. Clin. Invest. 81, 676; Buyon et al., 1988 J. Immunol. 140, 3156). Factors that have been reported to induce neutrophil activation include IL~
GM-CSF, G-CSF, MIP-l, IL-8 (IL-8 = interleukin-8, GM-CSF
= granulocyte/monocyte-colony stimulating factor, G-CSF
= granulocyte-colony stimulating factor),~TNF~, the complement fragment C5a, the microbe-derived peptide 3~ formyl-Met-Leu-Phe and the lipid-like molecules leukotriene B4 (LTB4) and platelet activating factor W094/14973 PCT~S93/1262~
i 2~5 ~S~g ~ 6 (Fuortes and Nathan, 1992, in Molecular Basis of Oxidative Damaqe bY LeukocYtes Eds Jesaitis, A.J. and Dratz, E.A. (CRC Press) pp. 81-90). In addition, phorbol esters (e.g., phorbol 12-myristate 13-acetate; r s PMA) have been proposed as a potent class of synthetic lipid-like neuL~o~hil agonists. With the exception of PMA, these agonists are believed to activate neutrophils by binding receptors on their surface. Receptors that are occupied by agonist molecules are believed to 10 initiate within the neuLIG~hil a cA~cA~e of events that ultimately will result in the physiological changes that accompany neutrophil activation. This process is known as signal transduction. The lipid-like PMA is proposed to affect neutrophil activation by passing through the 15 plasma membrane at the cell surface and directly interacting with intracellular components (i.e., protein kinase) of the signal transduction machinery.
There exist two general classes of compounds that have been reported to down regulate the function of 20 neutrophils, and these com~ou.,ds have been shown to mitigate inflammation. One group of anti-inflammatory compounds has been ~v~Gsed to function as inhibitors of neutrophil activation, and presumably adhesion, by acting on components of the signal transduction 25 machinery. A second class of anti-inflammatory compounds has been ~LO~O-^~ to block n~LL~hil infiltration into inflammatory foci by acting as direct inhibitors of the adhesive receptors that mediate contact between neutrophils and the vascular 30 endothelium.
Many of the anti-inflammato~y compounds currently used as therapeutics, including prostaglandins, catecholamines, and a group of agents kno~n as non-steroidal anti-inflammatory drugs (NSAIDs), are 3~ believed to fall into the first category (Showell and Williams, 1989, in Immuno~harmacologY, eds. Gilman, S.

W094/14g73 215 2 5 9 g PCT~S93/12626 C. and Rogers, T. J. tTelford Press, NJ] pp 23-63). For example, the ~nhAnse~ adhesiveness observed for TNF~-activated n~uLL~hils has been Le~o~Led as associated with decreased levels of a mediator of signal transduction, cyclic AMP (cAMP). (See Nathan and Sanchez, 1990 JCB 111, 2171). Exposure of neutrophils to prostaglandins and catecholamines has been correlated with elevated levels of intracellular cyclic AMP
(Showell and Williams, 1989). While signal transduction inhibitors have been used extensively as anti-inflammatory therapeutic agents, they have been shown to have several disadvantages including poor efficacy in acute inflammatory conditions, lack of specificity and undesirable side-effects such as gastric or intestinal ulceration, disturbances in platelet and central nervous system function and changes in renal function (Insel, 1990 in The Pharmacoloaical Basis of Thera~eutics, eds. Gilman, A. G., Rall, T. W., Nies, A.
S., and Taylor, P. ~Pergamon, NY], 8th Ed., pp.
638-681).
Glucocorticoids have long been r~cogn;zed for their anti-inflammatory properties. Steroid induced inhibition of neutrophils has been L~U-Led for several neutrophil functions, including adherence (Clark et al., 1979 Blood 53, 633-641; Ma~GL~o~, 1977 Ann. Intern.
Med. 86, 35-39). The mech~nisms by which glucocorticoids modulate ne~LLG~hil function are not well understood, but they are generally believed to involve the amplification or suppression of new proteins in treated neutrophils that play a key role in the inflammatory process (Knudsen et al., 1987 J. Immunol.
139, 4129). In particular, a group of proteins known as lipocortins, whose expression is induced~in neutrophils by glucocorticoids, has been associated with anti-inflammatory properties (Flower, 1989 Br. J.
Pharmacol. 94, 987-1015). Lipocortins may exert WO94/14973 , PCT~S931126?' .~, ~ 8 anti-neu~-G~hil effects by interacting with sites on the neutrophil surface (Camussi et al., 1990 J. Exp. Med.
171, 913-927), but there is no evidence to suggest that the li~o~GLLins act by directly blo~ki~ adhesive S proteins on the neutrophil. Apart from their beneficial anti-inflammatory properties, gl~ov~ Licoids have been associated with significant side-effects. These include suppression of pituitary-adrenal function, fluid and electrolyte disturh~nres, hypertension, hyperglycemia, glycosuria, susceptibility to infection, ulcers, osteoporos~s, myopathy, arrest of growth and behavioral disturbances (Insel, 1990).
A second class of anti-inflammatory compounds which are reported as direct inhibitors of neuLlu~hil adhesion to the vascular endothelium are monoclonal ant; hoA; es.
Monoclonal antibodies that re~oqn;ze and block ligand-binding functions of some of these adhesive molecules have been ~e~Gl ~ed to act as n vivo inhibitors of neutrophil-mediated inflammation. In particular, monoclonal an~; ho~; es to the CD18 subunit of the CD18 integrin complexes (i.e., CDlla/CD18, CDllb/CD18 and CDllc/CD18) on the surface of neutrophils have been r e~ ed to prevent a variety of neutrophil-mediated t; Se~l~ injury in animal models, including pulmonary edema induced by ,e~e-f~sion (Horgan et al, 1990 Am. J. Physiol. 259, L315-L319), organ injury induced by hemorrhagic shock (Mileski et al, l990 Surgery 108, 206-212), myocardial damage following ischemia/reperfusion (Winquist et al, 1990 Circulation III-701), edema and tissue damage following ischemia/reperfusion of the ear (Vedder et al, 1990 Proc. Natl. Acad. Sci.(USA) 87, 2643-2646), brain edema and death produced by bacterial meningiti~s (Tuomanen et al, 1989 J. Exp. Med. 170, 959-968), vascular injury and death in endotoxic shock (Thomas et al, 1991 FASEB J. 5, WO94/14973 PCT~S93/12626 2 15259g A509) and indomethacin-;n~llce~ gastric injury (Wallace et al, 1991 Gastroenterology 100, 878-883).
Monoclonal antibodies directed to the CDllb subunit have been reported by Todd, R.F. et al., U.S. Patent No.

4,840,793 (June 20, 1989), Todd, R.F. et al., U.S.
Patent No. 4,935,234 (June 19, 1990), Schlossman, S.F.
et al., U.S. Patent No. 5,019,648 (May 28, 1991) and Rusche, J.R. et al., International Application No. WO
92/11870 (July 23, 1992). Monclonal antibodies directed to CD18 subunit have been reported by Arfors, K.E., U.S.
Patent No. ~,797,277 (January 10, 1989), Wright, S.D. et al., European Patent Application No. 346,078 (December 13, 1989), Law, M. et al., European Patent Application No. 438,312 (July 24, 1991), Law, M. et al., European Patent Application No. 440,351 (August 7, 1991), Wright, S.D. et al., U.S. Patent No. 5,147,637 (September lS, 1992) and Wegner, C.D. et al., European Patent Application No. 507,187 (October 7, 1992).
Antibodies to other adhesive molecules have also been reported to have anti-inflammatory properties.
Monoclonal antibodies that ~e~Gyl.ize the counter-receptor of CDlla/CD18 and CDllb/CD18, ICAM-1 have been reported to prolong cardiac allograft survival (Flavin et al, 1991 Transplant. Proc. 23, 533-534) and prevent chemically in~l~c~ lung inflammation (Barton et al, 1989 J. Immunol. 143, 1278-1282). Furthermore, anti-selectin monoclonal antiho~ies have also been reported as active in animal models of neutrophil-mediated inflammation. Monoclonal antibodies to L-selectin have been reported to prevent neutrophil emigration into inflamed skin (Lewinshon et al., 1987 J.
- Immunol. 138, 4313) and inflamed ascites (Jutila et al., 1989 J. Immunol. 143, 3318; Watson et a~;, 1991 Nature 349, 164). Reports have also described inhibition of neutrophil influx into inflamed lung tissue by anti E-selectin monoclonal antibodies (Mulligan et al., 1991 WO94/14973 PCT~S93/lt626 2~5~9 lo J. Clin. Invest. 88, 1396; Gundel et al., 1991 J. Clin.
Invest. 88, 1407). While monoclonal antibodies to adhesive proteins have demonstrated the feasibility of using neutrophil adhesion inhibitors as anti-inflammatory agents, their utility as therapeutics requires further evaluation.
Soluble adhesive receptors obtAine~ by genetic engineering have been ~o~.-~~ as anti-inflammatory compounds. Soluble receptors, in which the transmembrane and intracellular domains have been deleted by rçcombinant DNA technology, have been tested as inhibitors of neutrophil adhesion to endothelial cells. The functional use of recombinant soluble adhesive molecules has been reported using CDllb/CD18 (Dana et al., 1991 Proc. Natl. Acad. Sci.(USA) 88, 3106-3110) and L-selectin (Watson et al., 1991).
Recently, a new class of anti-leukocyte compounds collectively termed "leumedins" has been reported.
These compounds have been LepO~ Led to block the recruitment n vivo of T lymphocytes and neuL o~hils into inflammatory lesions. The meC~nism of action o f the leumedins is unclear, but there is evidence that they do not function by blocking neu~ro~hil activation (Burch et al., 1991 Proc. Natl. Acad. Sci.(USA) 88, 355). It remains to be determined if leumedins block neutrophil infiltration by direct interference with adhesive molecules.
It has been suggested that parasites survive in their host by modulating host immunity and inflammatory response though the mechanisms by which this occurs remains unclear (Leid, W.S., 1987, Veterinary Parasitology, 25: 147). In this regard, parasite-induced immunosuppression in rod~nt models has been proposed (Soulsby et al., 1987, Immunol Lett. 16, 315-320). The various aspects of the modulation of host immunity by helminth parasites to evade immunological WOg4/14973 21 S 2 5g 9 PCT~S93/12626 `

attack has recently been reviewed. See Naizels et al.
(1993), Nature, 365:797-805.
Various parasites have been reported to have an affect on neutrophils of their host. For example, a protein isolated from the cestode, Taenia taeniaeformis, has been reported to inhibit chemotaxis and chemokinesis of equine neuLLophils, as well as inhibit neutrophil aggregation (C. Suquet et al., 1984, Int'l J.
Parasitol., 14: 165; Leid, R.W. et al., 1987, Parasite Immunology, 9: 195; and Leid, R.W. et al., 1987, Int'l J. Parasitol., 17: 1349). Peritoneal neu~lo~hils from mice infected with the cestode, Echinococcus multiocularis, have been reported to lose their ability to migrate toward parasite antigens and nonspecific chemoattractants with increasing time of infection (Alkarmi, T. et al., Exptl. Parasitol., 1989, 69: 16).
The nematode, Trichinella s~iralis, has been reported to either excrete and/or secrete factors which inhibit chemotaxis and p-nitroblue tetrazolium reduction (i.e., release of oxidative metabolites) but enhance chemokinesis of human neutrophils (Bruschi, F. et al., 1989, Wiadomosci Parazytologiczne, 35: 391). The sera of humans infected with the nematode, Trichinella s~iraLis, has been Le~O' ~ed to inhibit leukocyte chemotaxis and phagocytosis (Bruschi, F. et al., 1990, J. Parasitol., 76: 577). The saliva of the tick, Ixodes dammini, has been reported to inhibit neutrophil function (~iheiro et al, 1990, Exp. Parasitol., 70, 382). A protein secreted by the cestode, Echinococcus 30 qranulosus, has been reported to inhibit human neutrophil chemotaxis (Shepard, J.C. et al., 1991, Mol.
Biochem. Parasitol., 44: 81).
Another component of the host defen~e mechanism against invading pathogens are eosinophils.
Functionally, eosinophils are similar to neutrophils in that both cell types have the ability to phagocytose and W094/14973 PCT~S93/12626 ~9g 12 to release compounds that are either directly or indirectly toxic to pathogenic organisms. Eosinophils are distinguished from neutrophils by their morphologic features, constituents, products and associations with specific diseases. Although eosinophils have been reported to be capable of killing bacteria n vitro, this class of leukocyte alone is not believed sufficient to defend against bacterial infections n vivo.
Instead, it is thought that eosinophils afford primary defense against large organisms such as helminthic parasites (Butterworth AE, 1984; Adv. Parasitol.
23:143-235). Also, it is widely held that eosinophils can play a major role in certain inflammatory ~ic~s~c.
Specifically, subs~AnG~s released from eosinophils that are known collectively as cationic granule proteins, including major basic protein, eosinophil cationic protein and eosinophil-derived neurotoxin, have been implicated in asthma (Gleich GJ and Adolphson, CR, 1986;
Adv. Immunol. 39:177-253), inflammatory bowel disease (Hallren, R, 1989; Am. J. Med. 86:56-64) and atopic dermatitis (Tsuda, S, et al, 1992; J. Dermatol.
19:208-213). Moreover, other eosinophil products such as superoxide anions, hydroxyl radicals and singlet oxygen may also be involved in damage to host tissue in inflammatory disease states (Petreccia, DC et al, 1987, J. Leukoc. Biol. 41:283-288; Kanofsky, JR et al, 1988;
J. Biol. Chem. 263:9692-9696).
An early step in eosinophil-mediated inflammatory disease is believed to be the movement of eosinophils from the vascular compartment to tissue. The first step in this extravasation process is ~e~o~ed to be the adherence of eosinophils to the luminal surface of the vascular endothelium. Although mech~nis~s of eosinophil-endothelial cell adhesion are not as well defined as those involving adhesion by neutrophils, it is reported that members of the CD11/CD18 family of WO94/14973 PCT~S93/12626 2 1525~g integrins on the surface of the eosinophil are involved in eosinophil-endothelial adhesion (Lamas, AM, et al, 1988; J. Immunol. 140:1500; Walsh, GM, et al, 1990;
Immunology 71:258), and it is reported that the endothelial cell counter-receptor is likely ICAM-l (Wegner, CD, et al, 1990; Science 247:456-459). A
second integrin known as VLA-4 (very late antigen-4, a4bl) that is present on eosinophils, lymphocytes and monocytes but not neutrophils, is thought to contribute to eosinophil adherence by binding to the VCAM-l (vascular cell adhesion molecule-1) that is expressed on the surface of endothelial cells (Dobrina, A, et al, 1991, J. Clin. Invest. 88:20). IL-1 treatment of the endothelial cell monolayers has been ~epolLed to induce an increased adhesiveness for human basophils, eosinophils and neutrophils but treatment of these endothelial cells with an antibody directed to VACM-l was reported to inhibit both b~s~il and eosinophil adhesion but not neuL~o~hil adhesion. It has also been reported that monoclonal antibodies against VCAM-l inhibit lymphocyte and monocyte cell adhesion to stimulated endothelium (Carlos et al. (1990), Blood, 76:965-970; Rice et al., J. Exp. Med. (1990), 171:1369-1374) but not to neutrophils.
Approaches to the treatment of eosinophil-mediated inflammation have been similar to those adopted for neutrophil-mediated disease. For example, potential therapeutics under investigation for eosinophil-mediated inflammation include glucocorticoids (Evans, PM, et al, 30 1993, J. Allergy Clin. Immunol. 91:643-650). As is the case for other agents that have been reported to - modulate neutrophil function, these agents have been found to be sub-optimal in that they are~relatively non-specific and toxic. A second approach to anti-eosinophil therapy has been the use of compounds that directly inhibit the adhesion of eosinophils to W094/14973 PCT~S93112626 vascular endothelium. It has been reported that in animal models of asthma, monoclonal antibody against ICAM-1 blocks eosinophil infiltration into tissues.
Wegner et al. (1990), Science, 247:456-459. ICAM-1 and functional derivatives thereof have been proposed as anti-inflammatory agents. Anderson et al., European Patent Application No. 314,863 (April 29, 1988); Wegner et al., International Application No. W0 90/10453 (September 20, 1990).
However, there remains a need for potent, highly specific inhibitors of neutrophil and eosinophil function, in particular, adhesion to vascular endothelium, as a treatment for abnormal granulocyte-mediated inflammation. The present invention describes potent and specific inhibitors of neu~Lu~hil and eosinophil activity, in particular the adhesion of these granulocytes to vascular endothelial cells, derived from hookworms (such as AncYlostoma caninum) and related species.

SummarY of the Invention Among other factors, the present invention is based on our finding that the Neu~L~hil Inhibitory Factor of the present invention represents a pioneering step toward the development of a new generation of anti-inflammatory therapeutic prod~cts. This discovery will enable therapy for inflammatory disease based entirely on specific inhibition of the inflammatory response. The therapeutic advantages of this novel approach are realized through the specificity of Neutrophil Inhibitory Factor compared to current clinical treatment modalities such as steroids, catecholamines, prostaglAn~;nc, and non~teroidal anti-inflammatory agents. The currently used therapeutic agents demonstrate poor efficacy and multiple adverse reactions due to generalized systemic WO94/14973 PCT~S93/12626 effects that non-specifically target numerous biological processes in addition to the inflammatory process.
Nonetheless, the existence of this extensive panel of anti-inflammatory agents, although suboptimal, and the total funds ~Ypen~ed by the pharmaceutical industry in research in this area point to significant medical needs for effective anti-inflammatory agents and suggests that the novel and highly specific NeuL~ophil Inhibitory Factors of the present invention have important applications.
As noted in the Bac~Lou,ld, the inflammatory response may result in clinical syndromes ranging from debilitating arthritis and asthma to life threatening shock. In view of the severity of these disorders, the vast number of individuals afflicted therewith and the lack of suitable therapeutic intervention, the need for a breakthrough therapy re~L~~ents a long felt need which has not been met. The Neu~.~hil Inhibitory Factor of the present invention is believed to meet this need by providing the potential for a lifesaving therapy which is currently being sought throughout the international - medical and pharmaceutical research communities.
Further, in view of the myriad conditions associated with undesired and/or abnormal inflammatory conditions which appear to be associated with neutrophil activity, there remains a need for potent, highly specific inhibitors of neu-Lo~hil function, in particular, adhesion to vascular endothelium, as a treatment for abnormal neutrophil-mediated inflammation.
The present invention is believed to fulfill this need by disclosing a potent and specific inhibitor of neutrophil activity, in particular the adhesion of neutrophils to vascular endothelial cell~, derived from hookworms (such as Ancvlostoma caninum) and related species.

W094/14973 PCT~S93/12626 The present invention is directed to a neutrophil inhibitory factor ("NeuL~o~hil Inhibitory Factor" or "NIF") which may be isolated from natural sources or made by recombinant methods. Neutrophil Inhibitory Factor is a protein which is neither an antibody, a member of the integrin or selectin families nor a member of the immunoglobulin superfamily of adhesive proteins and which when isolated from a parasitic worm is a glycoprotein. Recombinant NIF produced by certain expression systems may or may not be glycosylated, or may be glycosylated to a variable degree. However, such NIFs whether glycosylated or not are considered to be within the scope of the present invention.
In one aspect, the present invention provides NIFs which contain, as part of their total amino acid sequence, an amino acid sequence selected from the group consisting of (a) Arg-X~-X2-Phe-Leu-X3-X4-His-Asn-Gly-Tyr-Arg-Ser-X5-Leu-Ala-Leu-Gly-His-X6-X7-Ile, wherein X1 is Leu or Arg; X2 is Gln, Lys or Arg; X3 iS Ala or Arg; X4 iS Leu or Met; X5 iS Lys, Arg, Leu or Ile; X6 is Val or Ile; and X7 is Ser, Gly or Asn;
(b) Ala-X8-X9-Ala-Ser-X1O-Met-Arg-Xl~-Leu-Xl2-Tyr-Asp-Cys-X~3-Ala-Glu-XI4-Ser-Ala-Tyr-Xl5-Ser-Ala, wherein X8 is His or Pro; X9 iS Thr, Arg or Ser; X~O is Arg or Lys;
X~ is Ile or Tyr; X~2 is Asp, Lys or Glu; Xl3 is Asp or Glu; Xl4 is Gly, Lys or Arg; and X~5 iS Glu, Met, Thr or Val;
(c) Ser-X~6-Phe-Ala-Asn-XI7-Ala-Trp-Asp-X~8-Arg-Glu-Lys-X~9-Gly-Cys-Ala-Val-Val-X20-Cys, wherein X~6 is Asn or Asp; X1~ is Val or Leu; X18 iS Ala or Thr; X19 iS Leu, Val or Phe; and X20 iS Thr, Lys or Asn;
(d) His-Val-Val-Cys-His-X2l-X22-Pro~Lys, wherein X
is Tyr or Ile; X22 is Gly or no residue;
(e) Ile-Tyr-x23-x24-Gly-x2s-pro-cys-x26-x27-cys-x2s-X29-Tyr, wherein X23 iS Thr, Ser, Lys or Glu; X24 is Thr, W094114973 PCT~S93/12626 Val or Ile; X25 is Val, Lys or Thr; X26 is Arg, Ser or Asp; X27 iS Asn, Gly, Asp or Arg; X28 iS Asn, Ser or Thr;
and X29 iS Gly, Glu or Asp; and (f) Cys-X30-X31-Asp-X32-Gly-Val-Cys-X33-Ile, wherein X30 is His, Ile or Asn; X3~ iS Ala, Pro or Asp; X32 is Glu, Val, Asp or Ile; X33 iS Ile, Val or Phe. Such NIFs exhibit neutrophil inhibitory activity.
In another aspect, the present invention provides mutant NIFs wherein certain asparagine residues are replaced with glutamine residues which is-believed to result in tX~ reduced glycosylation of these NIFs. The mutant NIFs contain, as part of their total amino acid sequence, an amino acid sequence selected from the group consisting of peptides (a) to (f) hereinabove. Such NIFs exhibit ne~L~ophil inhibitory activity.
In another aspect, the present invention provides NIFs which contain, as part of their total amino acid sequence, an amino acid sequence enco~eA by a nucleic acid sequence which is sufficiently complementary to hybridize to certain nucleic acid probes. Such NIFs exhibit neutrophil inhibitory activity. The present invention includes within its scope these nucleic acid probes.
In another aspect, the present invention provides nucleic acid molecules encoding for a NIF and which are isolated as described herein. Such isolated nucleic acid molecules include expression vectors containing a nucleic acid sequence ~nCoAi~g a NIF. The present invention also includes the host cells transformed by such expression vectors.
In another aspect, the present invention provides methods for making biologically active NIFs, wherein such NIFs are expressed and, optionally, secreted. The present invention also includes the NIFs made by these methods.

W094/14973 PCT~S93/12626 2~5g~ 18 In another aspect, the present invention provides methods of making NIFs comprising preparing a cDNA
library from a source suspected of having a NIF and hybridizing certain oligonucleotide probes of the present invention to the nucleic acid molecules from the source. Such NIFs exhibit neutrophil inhibitory activity. The present invention also includes the NIFs made by these methods.
In another aspect, the present invention provides methods of detecting in a sample the presence of a nucleic aci~ molecule ~n~oAing a NIF, which methods comprise the combining the sample thought to contain such nucleic acid molecule with a probe of the present invention and detecting the presence of hybridized probe.
In another aspect, the present invention provides monoclonal ant; hoA; es which bind to NIF. The present invention also includes the hybridoma cell lines which make such an~; hoA; es, a method of purifying NIF using such monoclonal antibodies and method of detecting in a sample the presence of NIF using such anti hoA; es.
In another aspect, the present invention provides a method for detecting in a sample NIF mimics which compete with NIFs for binding to the CDllb/CDl8 receptor, which method comprises contacting a sample with the CDllb/CDl8 receptor. Also provided is a method for detecting in a sample NIF mimics which compete with NIFs for binding to the I-domain portion of the CDllb/CDl8 receptor, which method comprises contacting a sample with a recombinant peptide comprising the I-domain of CDllb/CDl8 receptor. Such NIF mimics exhibit neutrophil inhibitory activity. The preçent invention also includes the NIF mimics detected b~ these methods.

In another aspect, the present invention provides a method for detecting in a sample a NIF antagonist which W094l14973 PCT~S93/12626 prevents NIF b;n~i~g to the CDllb/CD18 receptor, which method comprises contacting such sample with the CDllb/CD18 receptor. Also provided is a method for detecting in a sample NIF antagonist which compete with NIFs for binding to the I-domain portion of the CDllb/CD18 receptor, which method comprises contacting a sample with a recombinant peptide comprising the I-domain of CDllb/CD18 receptor. Such NIF antagonists do not exhibit neutrophil inhibitory activity themselves.
The present invention also includes the NIF antagonists detected by~these methods.
In another aspect, the present invention provides methods of using NIF to treat inflammatory conditions, especially to prevent or decrease inflammatory responses, which methods comprise administering to a mammal a therapeutically effective amount of NIF.
Other features and advantages of the present invention will be apparent from the following descriptions of the preferred embodiments and from the claims.

Definitions In accordance with the present invention and as used herein, the following terms are defined with the following me~n;ngs, unless explicitly stated otherwise.
The term "amino acid" refers to the natural L-amino acids. The natural amino acids shall be referred to by their names or may be abbreviated as shown below:
L-amino acid Abbreviation Three letter One Letter 30 alanine.............. Ala............ A
arginine............ Arg............ R
asparagine.......... Asn............ N
aspartic acid....... Asp............ D
cysteine............ Cys............ C
35 glutamine............ Gln............ Q

WO94/14973 PCT~S93112626 59g glutamic acid........ Glu............ E
glycine.............. Gly............ G
histidine............ His............ H
isoleucine........... Ile............ I

5 leucine............... Leu............ L
lysine............... Lys............ K
methionine........... Met............ M
phenylalanine........ Phe............ F
proline.............. Pro............ P
lO serine................ Ser............ S
threonine... ~............ Thr............ T
tryptophan........... .... Trp............ W
tyrosine............. .... Tyr............ Y
valine............... .... Val............ V

The term "amino acid residue" refers to -NH-CH(R)-C0-, wherein R is the side chain group disting~l; Ch i ng each amino acid. For cyclic amino acids, the residue is (CH2)~
< ~~
N

wherein x is l, 2 or 3 representing the azetidine-carboxylic acid, proline or pipecolic acid residues, respectively.
The term "isoform" refers to a family of related - proteins from a single organism having homologous sequences of amino acid residues interspersed with variable sequences.
The term "nucleic acid" refers to polymers of either deoxynucleic acids or ribonucleic~acids, either single-stranded or double-stranded.
The term "isolated nucleic acid" refers to nucleic acids which are isolated by biochemical or molecular W094114973 PCT~S93/12626 - 21S2~99 biology t~chniaues such as centrifugation, chromatography, ele~L~ophoresis, hybridization and the like.
The term "NIF mimic" refers to a small molecule, peptide, peptide analog or protein, which competes with NIF for binding to the CDllb/CDl8 receptor or the I-domain portion of the CDllb/CDl8 recptor. A NIF mimic is also characterized as having neutrophil inhibitory activity, eosinophil inhibitory activity or both such activities.

The term "NIF antagonist" refers to a small molecule, peptide, peptide analog or protein, which prevents the binding of NIF to the CDllb/CDl8 receptor or the I-domain portion of this receptor, and does not possess any significant neu~G~hil inhibitory activity.
A NIF antagonist prevents bin~i ng of NIF to the CDllb/
CDl8 receptor or the I-domain portion of the CDllb/CDl8 receptor, by binding to a site on NIF which is rea,uired for binding to the receptor in effect sterically hindering binding, or alternatively, by binding to a site on NIF which results in a conformational change to the site needed for such bin~g which change substantially weakens or abolishes bi nA; ng .

Brief Descri~tion of the Drawinas Figure l depicts a chromatogram of hookworm lysate obt~;ne~ as described in the Example 2(A) run on the Example 2(B) Concanavalin A SephArose column.
Figure 2 depicts a chromatogram of Concanavalin A-purified hookworm lysate run on the Example 2(C) ~ 30 Superdex 200 column.
Figure 3 depicts a chromatogram of~the Concanavalin A Sepharose/Superdex purified hookworm lysate run on the Example 2(D) ceramic hydroxyapatite column.

W094114973 PCT~S93112626 Figure 4 depicts a chromatogram from reverse phase HPLC of hookworm lysate isolated by Concanavalin A
Sepharose, Superdex 200 and hydroxyapatite chromatography as described in Example l(E).
Figure 5 depicts a gel pattern run using SDS-gel electrophoresis of the HPLC isolate and certain molecular weight st~n~rds.
Figure 6 depicts laser-desorption time-of-flight mass spectrometry of the purified Neutrophil Inhibitory Factor of the present invention.
Figure 7 depicts the amino acid sequence of proteolytic fragments prepared from Neutrophil Inhibitory Factor isolated from canine hookworms.
Figure 8 depicts the nucleotide sequence of the co~i~g region of NeuL~uphil Inhibitory Factor CDNA
(clone lFL) and its predicted amino acid sequences.
Figure 9 depicts the alignment of the predicted amino acid seguences of several NeuLLu~hil Inhibitory Factor isoform clones.
Figure lO depicts the anti-inflammatory effect of varied doses of Neutrophil Inhibitory Factor isolated from canine hookworms administered intraperitoneally in an animal model of inflammation.
Figure ll depicts the anti-inflammatory effect of Neutrophil Inhibitory Factor isolated from canine hookworms administered either intraperitoneally or intravenously in an animal model of inflammation.
Figure 12 depicts the anti-inflammatory effect of recombinant Neutrophil Inhibitory Factor produced in Pichia ~astoris administered n vivo in an animal model of inflammation.
Figure 13 depicts the effect of recombinant NIF on the inhibition of eosinophil adherence ~o TNF-stimulated HUVEC monolayers. Data points are means of triple determinations of one experiment.

WO94/14973 PCT~S93/12626 Figure 14 depicts genetic map of the expression ; vector Pma5-NIl/3. The vector contains the following elements: (i) a ColEl type origin of replication (ORI);
(ii) the intercistronic region of filamentous phage fl including the origin of replication (fl ORI); (iii) the beta-lactamase gene which confers resistance to ampicillin (bla); (iv) the chloramphenicol acetyl transferase gene which contains an amber translational stop codon as the result of a single nucleotide substitution (cat-am); (v) the phage lambda PR promoter;
(vi) a small leader cistron; (vii) the methionyl-NIF
encoding region and (viii) two tandemly arranged copies of the central transcription terminator of phage fd (fdT). The sequence of the Met-NIF expression cassette (blown-up region) is shown in Figure 15. The Pma/c family of vectors have been described. See Stanssens et al., Nucl. Acids Res. 17, 4441-4454, 1989.
Figure 15 depicts the nucleotide base sequence of the two-cistron Met-NIF expression cassette of PmaS-NIl/3. The encoded methionyl-NIF and leader peptide are shown in the one-letter code. The following features are indicated: the -35 and -10 regions of the phage lambda PR promoter, the transcription initiation point, the Shine-Dalgarno elements prPce~i~g the leader cistron (SDC~) and the Met-NIF gene (SD) and some restriction sites. The vector was obtained by ligation of the PCR amplified NIF-lFL coding region to the recipient vector which was cleaved by NcoI, treated with the Klenow fragment of DNA polymerase I and subsequently digested with HindIII (both ligation points are indicated). The construction scheme fuses the 5'-end of the NIF coding region to an ATG initiator codon.
Figure 16 depicts a comparison of ~he nucleotide and amino acid sequences of NIF proteins from hookworms.
The NIF-lFL nucleotide sequence is numbered; this numbering is also used to refer to positions in other WO94/14973 PCT~S93112626 ~ 24 genes. PCR-NIF7 and PCR-NIF20 were recovered by PCR-technology: the (regions matching with the) PCR-primers are italicized. AcaNIF7 and NIF-lFL9 differ at only one position (G to E replacement;
nucleotide-substitution located at position 647). The one remaining uncertainty in these sequences is at position 660 in PCR-NIF20. No poly(A~) tail was found in NIF-lFL sequence. Underlined sequences in the 3'-untranslated region (UAUAAA and AGUAAA) may serve as polyadenylation signals. Only the NIF-lFL, NIF-lFL9 and AcaNIF24 cD~As contain an entire secretion signal. The potential N-glycosylation sites (N-X-T/S) are underlined.
Figure 17 depicts a comparison of the potency of recombinant NIF-lFL with that of the recombinant proteins AcaNIF6 and AcaNIF24. The three purified proteins were tested in both the hy~Loyen peroxide release assay of Example l(E) (panel A) and the neutrophil-plastic adhesion assay of Example l(C) (panel B).
Figure 18 depicts a comparison of recombinant NIF-lFL with recombinant AcaNIF4 in the competition binding assay of Example l(F). Samples contA;~ing a fixed amount of biotinylated NIF-lFL and varying amounts of either AcaNIF4 or NIF-lFL, were assayed on immobilized LM2/Mac-l complex. The amount of bound biotinylated NIF-lFL was detected with ExtrAvidin conjugated with alkaline phosphatase.
Figure l9 depicts the nucleotide and amino acid sequence of a NIF protein from A. ceYlanicum (AceNIF3).
The underlined sequence (GAATTCCG) derives from the EcoRI linker that was added onto the CDNA. The sequence which may function as polyadenylation si~nal (AAUAAA) is also indicated. The encoded protein contains l0 potential N-glycosylation sites (N-X-T/S).

W094/14973 PCT~S93/12626 Figure 20 depicts the b;n~;~g of phages displaying a NIF protein from A. ceylanicum (AceNIF3) to Mac-l.
Wells coated with LM2/Mac-l complex were first incubated with varying amounts of Pichia-produced RNIFl (or buffer in the control experiment); after 30 minutes, 101 virions displaying either NIF-lFL or AceNIF3 were added and the incubation cont;~ A for another 90 minutes.
Retained phages were detected with rabbit anti-phage serum and goat anti-rabbit alkaline phosphatase conjugate. The various components were incubated in PBS
containing ~ Mm MgCl2, 1 Mm CaCl2 and 0.1% skim-milk (phage samples contained 1~ skim-milk). Unbound material was removed by washing with PBS containing 1 Mm CaCl2, 1 Mm MgCl2, 0.1% Tween 20 and 0.02% thimerosal.
The relative amount of bound phages was calculated by taking the OD~ reading obtained in the control experiment as 100%.
Figure 21 depicts the synthesis of functional NIF-lFL by PAN-NIF-lFL in either a phage-attached or 'soluble' form. The PAN-NIF-lFL vector contains the following elements:
1) a NIF-lFL contA;n;ng gene fusion which is placed under the transcriptional cGl-LLol of the Plac promoter.
The gene fusion consists of the pelB secretion signal, the NIF-lFL coding region, and the filamentous phage M13 geneIII (gIII). Between the NIF-lFL and the GIII
sequences a TAG amber translational stop codon is present. The NcoI and NotI restriction sites used for the cloning of the NIF-lFL coding region are indicated.
2) the bla gene which confers resistance to 100~g/ml ampicillin or carbenicillin (bla/ApR).
3) the intergenic region, including the origin of replication, of filamentous phage M13 (~i3-IR/ORI).
4) a ColEl-type plasmid borne origin of replication (ColE1-ORI).

WO94tl4973 PCT~S93/12626 ~ 26 In su+ bacteria such as;TG1, the PAN-NIF-lFL
phagemid-vector codes for a NIF-lFL-pgIII fusion protein (pgiii; product of GIII) which becomes incorporated into filamentous virions upon infection of TGl~PAN-NIF-lFL]
cells with M13-VCS 'helper'-phage (Statagene). In su strain WK6, PAN-NIF-lFL directs the synthesis of 'free' (not phage-attached) RNIF1 which binds to the anti-NIF
MAb 3D2 and to Mac-1.
TG1: ~(lac-proAB), hsd 5 (rK~K-), thi, supE / F'[traD36, lacIq,lacZ~M15, proA+B+].
WK6: ~(lac-proAB), galE, strA / F'~lacIq,lacZ~M15, proA+B+].
Figure 22 depicts ~T.T,C~ with NIF-lFL-displaying phage. NIF-displaying phages (or non-displaying control phages, e.g. M13-VCS; in each experiment about 5x109 phage particles were added) were incl~hAted in microtiter wells coated with Mac-l (immunopurified on LM2-Sepharose), LM2/Mac-l, LM2 or the non-neutralizing anti-NIF Mab 3D2. In some experiments non-phage-attached 'soluble' NIF (sNIF) was allowed to react with the immobilized material (1 ~M) 30 minutes prior to addition of the phages. Bound phages were detected with a rabbit anti-M13 serum and an alkaline phosphatase conjugated goat anti-rabbit serum.
Figure 23 depicts a comparison of the potency of AcaNIF1 with that of the mutants, AcaNIFl/~h,G11-7 and AcaNIF1/~G11-5, in a neutrophil-plastic adhesion assay of Example l(C).
Figure 24 depicts binding of biotinylated rNIF to lymphocytes, monocytes and granulocytes as described in Example 30.
Figure 25 depicts direct concordance of NIF binding and CDllb/CD18 expression as determined~by dual fluorescence analysis flow cytometry of peripheral lymphocyte populations as described in Example 31.

W O 94/14973 PCTrUS93tl2626 21~2599 -Detailed Descri~tion of the Invention 1. Neutrophil Inhibitory Factor.
The present invention in its various aspects is directed to NeuLL~hil Inhibitory Factor ("NIF"), a protein that inhibits neuLLo~llil activity and which is not an antibody, an integrin, a selectin or a member of the immunoglobulin superfamily of adhesive proteins and which, when isolated from a parasitic worm, is a glycoprotein. Recombinant NIFs proAllce~ by certain expression systems are not glycosylated. Such non-glycosy~ated NIFs are considered to be within the scope of the invention.
The inhibition of neutrophil activity by the NIFs of the present invention includes but is not limited to inhibition of one or more of the following activities by neutrophils: release of hyd~Gyen peroxide, release of superoxide anion, release of myeloperoxidase, release of elastase, homotypic ne~LLo~hil ayyLeyation~ adhesion to plastic surfaces, adhesion to VA~ r endothelial cells, chemotaxis, transmigration across a monolayer of endothelial cells and phagocytosis. Preferred assays include those where inhibition of neutrophil activity is demonstrated by an n vitro assay which determines adhesion of ne~LLo~hils to v~ r endothelial cells, release of hydLoyen peroxide from neuLLopllils, homotypic neutrophil aggregation or adhesion of neutrophils to plastic surfaces. Preferred NIFs would have an IC50 of about 500 Nm or less, more preferably less than 100 Nm, as measured by one of these neuL,o~hil activity assays.
An IC50 is that concentration of a NIF giving 50%
inhibition of the measured activity (see Example 1).
The NIFs of the present invention are further characterized as also having the abilit~ to bind to the CDllb/CD18 receptor (see Example 14). Preferred assays for determining the binding of NIF to CDllb/CD18 is described in Example l(F).

W094/14973 PCT~S93/12626 9~ 28 The NIFs of the present invention are further characterized as also having the ability to bind to the I-domain portion of the CDllb/CD18 receptor (see Example 32). A preferred assay for determining the binding of the NIF to the I-domain portion is described in Example 32.
The NIFs of the present invention may be further characterized as having eosinophil inhibitory activity.
A preferred assay for determining eosinophil inhibitory activity is the inhibition of eosinophil activity demonstrate~ by an n vitro assay which determines adhesion of neutrophils to vascular endothelial cells as described in Example 29. A preferred NIF would have an - IC50 of about 500 Nm or less, more preferably less than 100 Nm, as measured by this eosinophil activity assay.

(a) Enriched Compositions.
In another aspect, the present invention is directed to compositions enriched for NIF, comprising NIF and which are a isolated by chromatographic or molecular biology methods, or a combination of both methods, from a parasitic worm, preferably a nematode.
Suitable parasitic worms include those selected from species of the phyla Platyhelminthes, Nematoda, Nematomorpha or Acanthoceph~la. An especially preferred source is endoparasitic hookworm species, such as those found to infect canines. It is believed that certain isoforms of NIF are produced by canine hookworm Ancylostoma species such as AncYlostoma caninum.
Another suitable source is the endoparasitic worm species Toxocara canis. Substantially similar compounds may be isolated from other nematode species, as well as from other endoparasites of other phyla~ Preferred sources for NIF include parasites, including parasitic worms, particularly endoparasitic nematodes and especially hookworm species, including AncYlostoma .

W094/14g73 PCT~S93/12626 ~152599 braziliense, AncYlostoma caninum, AncYlostoma ceylanicum, Ancylostoma duodenale, Ancylostoma ia~onica, AncYlostoma malaYanum, AncYlostoma tubaeforme, Bunostomum ~hlebotomum, Cyclodontostomum ~urvisi, Necator americanus, Necator arqentinus, Necator suillus, and Uncinaria stenoce~Ala.
The enriched compositions may be enriched for NIF
in one aspect by chromatographic methods, which methods may include chromatography on Concanavalin A æ~phArOse hydroxyapatite or an anion eY~hAnge column, gel filtration chromatography preferably using Superdex0 200, C4 reverse phase HPLC, isoelectric focusing or a combination of those methods or equivalent methods used for separating proteins or proteinaceous factors. For example, in place of Concanavalin A, other immobilized lectins may be used. In place of Superdex0 200, other acrylamide- or agarose-based gel filtration media which fractionate proteins in the a~lop~iate molecular weight range may be used; these include those sold under the tradenames, Sephacryl~ and Superose~ (Pharmacia).
According to a preferred embodiment, the enriched composition is comprised of NIF. The NIF therein is a glycoprotein derived from or isolated from a parasitic worm, preferably a nematode, and more preferably a hookworm species, especially a canine hookworm species or, alternatively, a Toxocara species, or a compound, preferably a protein, which is substantially similar to said glycoprotein. It is believed that certain isoforms of said glycoprotein are produced by the canine hookworm AncYlostoma caninum. By substantially similar is meant that the compound exhibits selective neuLLo~hil inhibitory activity similar to that of the glycoprotein, and, preferably has an IC50 of about 500~Nm or less, more preferably less than lO0 Nm, as measured by neutrophil activity assays such as those described herein.

WOg4/14973 PCT~S93112626 ~S~g , (B) Glyco~rotein NIF and Isoforms.
In another aspect, the NIFs of the present invention comprise a purified glycoprotein. A NIF may be determined to be a gly~o~LoLein by evaluating binding to Concanavalin A Sepharose (see Example 2(B)) and by positive testing as a glyco~lGLein in GlycoTrack~
diagnostic assay for the presence of carbohydrate groups (see Example 7).
One glycoprotein having neutrophil inhibitory activity which was isolated from canine hookworms has the followi~ characteristics: This glycG~LoLein is acidic and exhibits an isoelectric point of about 4.5 as determined by isoelectric focusing (see Example 3). It has an observed molecular weight of about 41,000 daltons (+ 3,000) as determined by laser-desorption time-of-flight mass spectrometry (see Example 6). Its behavior when subjected to SDS-polyacrylamide gel electrophoresis indicated that it contained multiple disulfide bonds, since the reduced glycoprotein migrated on the gel at a significantly higher apparent molecular weight (see Example 5). The glycoprotein was demonstrated to specifically inhibit neutrophil activity and not to act as a general cytotoxin in another cell adhesion assay (see Example 13). This gly~o~otein was demonstrated to inhibit neutrophil adhesion to vascular endothelial cells and homotypic neuLLo~hil aggregation.
One such enriched composition (see Example 2(D)) exhibited an IC50 of about l0 Nm. An IC50 is that concentration of inhibitor giving 50% inhibition of the measured activity (see Example l). This glycoprotein was demonstrated to inhibit peritoneal inflammatory response when administered intraperitoneally or intravenously in an animal model of acute inflammation (cée Example 16).
This enriched composition was demonstrated to inhibit hydrogen peroxide release from neutrophils (see Example l(E)) and neutrophil adhesion/spreading on plastic (see W094/14973 215 2 5 99 PCT~S93112626 -Example l(C)). The Example 2(D) preparation had an IC50 of about 10 Nm. An enriched composition of the neutrophil function inhibitory factor was shown to have 2 no inhibitory effect on platelet aggregation (see 5 Example 13).
A second glycoprotein having neuLLo~llil inhibitory activity has been isolated from Toxocara canis. This glycoprotein has an observed molec~ r weight of about 20,000 daltons as determined by moleclllAr sieve 10 chromatography. This glycoprotein was demonstrated to inhibit neu~rophil adhesion to vascular endothelial cells and neutrophil adhesion/spreading on plastic.
In another aspect, the present invention is directed to methods for making enriched compositions 15 comprising NIF, wherein such compositions are isolated from natural sources which may include but are not limited to the parasitic worms.
One preferred embodiment comprises isolating these enriched compositions by chromatographic methods, which 20 methods would include chromatography on Concanavalin A-Sepharose0, hydroxyapatite or a~ anion exchange column, gel filtration chromatography preferably using Superdex~
200, C4 reverse phase HPLC, isoelectric focusing or a combination of those methods or equivalent methods used 25 for separating proteins or proteinaceous factors. For example, in place of Concanavalin A-, other immobilized lectins may be used. In place of Superdex~ 200, other acrylamide- or agarose-based gel filtration media which fractionate proteins in the a~o~iate molecular weight 30 range may be used; these include those sold under the tradenames, Sephacryl~ and Superose~ (Pharmacia).
Preferred methods of preparing enriched compositions comprising NIF are provided which compr~se subjecting a lysate from a parasitic worm to the following isolation 35 steps (a) chromatography on Concavalin-A Sepharose~, and (b) gel filtration on Superdex~ 200, and (c) WO94/14973 PCT~S93/12626 ~95 chromatography on ceramic hydroxyapatite. The enriched composition may be further enriched for NIF subjecting it to the further isolation step of reverse phase high performance liquid chromatography (HPLC) using a C4 column. Examples of methods of preparing the enriched compositions according to the preæent invention are described in Examples 2 to 5.
The enriched compositions comprising NIF isolated by chromatographic methods are at least about 50% pure, that is, they contain at least about 50% NIF.
Preferably,~the composition is enriched at least about 200-fold. According to another preferred embodiment, substantially pure NeuL~hil Inhibitory Factor is prepared. By "substantially pure" is meant at least about 90 percent pure. More preferably the NeuLLo~hil Inhibitory Factor so prepared is chromatographically pure.
It is believed that a NIF isolated from a particular source may include multiple isoforms.
Accordingly such isoforms are considered to be scope of the present invention. Accordingly, in another aspect, the present invention is directed to a NIF which includes an amino acid sequence selected from the group consisting of (a) Arg-X~-X2-Phe-Leu-X3-X4-His-Asn-Gly-Tyr-Arg-Ser-X5-Leu-Ala-Leu-Gly-His-X5-X7-Ile, wherein Xl is Leu or Arg; X2 iS Gln, Lys or Arg; X3 iS Ala or Arg; X4 iS Leu or Met; X5 is Lys, Arg, Leu or Ile; X6 is Val or Ile; and X7 is Ser, Gly or Asn;
(b) Ala-X8-Xg-Ala-Ser-X~O-Met-Arg-X~-Leu-X~2-Tyr-Asp-Cys-X~3-Ala-Glu-X~4-Ser-Ala-Tyr-X~5-Ser-Ala, wherein X8 is His or Pro; Xg is Thr, Arg or Ser; X~0 is Arg or Lys;
X1l is Ile or Tyr; X~2 is Asp, Lys or Gluj X13 iS Asp or Glu; X~4 iS Gly, Lys or Arg; and X~5 is Glu, Met, Thr or Val;

WO94/14973 PCT~S93/12626 2IS~S~9 (c) Ser-XI6-Phe-Ala-Asn-X~7-Ala-Trp-Asp-XI8-Arg-Glu-Lys-X~9-Gly-Cys-Ala-Val-Val-X20-Cys, wherein X~6 is Asn or Asp; X17 is Val or Leu; X18 is Ala or Thr; X~9 is Leu, Val or Phe; and X20 iS Thr, Lys or Asn;
(d) His-Val-Val-Cys-His-X2l-Xn-Pro-Lys, wherein X
is Tyr or Ile; Xn is Gly or no residue;
(e) Ile-Tyr-X~-X~-Gly-X~-Pro-Cys-X26-Xn-Cys-X28-X29-Tyr, wherein X~ is Thr, Ser, Lys or Glu; X~ is Thr, Val or Ile; X~ is Val, Lys or Thr; X26 is Arg, Ser or Asp; X~ is Asn, Gly, Asp or Arg; X28 iS Asn, Ser or Thr;
and X~ is G~y, Glu or Asp; and (f) Cys-X30-X3l-Asp-X32-Gly-Val-Cys-X33-Ile, wherein X30 is His, Ile or Asn; X3l is Ala, Pro or Asp; X32 is Glu, Val, Asp or Ile; X33 iS Ile, Val or Phe.
The NIF may include from 1 to 6 of amino acid sequence (a) to (f) above. Preferably the NIF includes all of amino acid sequences (a) to (f). Preferably, the listed amino acid sequences appear in the following order in the protein (from amino terminal end to carboxy terminal end): (a), (b), (c), (d), (e), (f). Additional amino acid residues or peptide sequences may be interspersed between the above sequences or may be located at the amino terminal and/or carboxy terminal end of the protein (for example, see Figures 7 and 8). The amino acid sequences of some of the NIF isoforms containing one or more of (a), (b), (c), (d), (e) or (f) are shown in Figures 9 for canine hookworm NIF, in Figure 16 for AncYlostoma caninum NIF and in Figure 19 for AncYlostoma ceYlanicum NIF. Preferred are NIFs comprising the amino acid sequence depicted in Figure 8.
According to the present invention, included are NIFs which have one or more of amino acid sequences (a), (b), (c), (d), (e) and (f) and exhibit neutrophil inhibitory activity in at least one of the in vitro assay. Suitable assays include those assays which determine adhesion of neutrophils to vascular WOg4/14973 PCT~S93/12626 endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces. Preferred are NIFs which have an IC50 of about 500 Nm or less, more preferably less than lOO Nm, as measured by one these neutrophil activity assays and which do not substantially inhibit platelet aggregation at such neutrophil inhibitory concentrations. An IC50 is that concentration of a NIF giving 50% inhibition of the measured activity (see Example 1).
The NIFs of the present invention are further characterized as also having the ability to bind to the CDllb/CD18 receptor (see Example 14). Preferred assays for determining the binAin~ of NIF to CDllb/CD18 receptor are described in Example l(F).
The NIFs of the present invention are further characterized as also having the ability to bind to the I-domain portion of the CDllb/CD18 receptor (See Example 32). A preferred assay for determining the binding of NIF to the I-domain portion is described in Example 32.
The NIFs of the present invention are also further characterized as having eosinophil inhibitory activity.
A preferred assay for determining eosinophil inhibitory activity is the inhibition of eosinophil activity demonstrated by an n vitro assay which determines adhesion of neutrophils to vascular endothelial cells as described in Example 29. Preferred are NIFs having an IC50 f about 500 Nm or less, more preferably less than 100 Nm, as measured by this eosinophil activity assay.

(C) Recombinant NIFs.
In another aspect, the present invention is directed to NIFs made by methods compri~ing hybridizing the nucleic acid molecules from a source suspected to contain a NIF to certain oligonucleotide primers or CDNA
made from such primers. Such NIFs exhibit neutrophil ~WO94/14973 PCT~S93/12626 21~599 inhibitory activity. In yet another aspect, the present invention is directed to these methods of making NIFs.
In one preferred aspect, the present invention is directed to NIFs comprising an amino acid sequence which is encoded by a nucleic acid sequence which is sufficiently complementary to hybridize to a primer derived from the amino acid sequence of a NIF.
Preferred in the PCR cloning method are single stranded DNA primers of 20-lO0 nucleotides derived from the sequence of NIF from AncYlostoma canium. Preferred are primers having the following characteristics: limited degeneracy; adherence to codon usage preferences of the particular species from which the library is constructed and primers that target seq~lPnc~c which are concerved among the twelve AncYlostoma caninum NIF isoforms. Each PCR reaction utilizes two primers: a 5-primer that corresponds to the sense strand and a 3'-primer that corresponds to the antisense strand of the NIF coding sequence. Especially preferred are the primers 5-CTCGAATTCT(GATC)GC(ATC)AT(ATC)(CT)T(GATC)GG(ATC)TGGGC-3' and 5'-CTCGAATTCTT(TC)TCTGG(GA)AA(GA)CG(GA)TC(GA)AA-3'.
The nucleotides within enclosing parentheses are redundant in that any one nucleotide may be used at the position enclosed by such parentheses.
The nucleic acid sequence of the DNA primers are preferably derived from the sequence of NIF from Ancylostoma canium. As described above, one example of NIF of the present invention comprises a glycoprotein which has been isolated in substantially pure form.
Using procedures known in the art, one of ordinary skill ~ in the art can use this protein to derive its amino acid sequence. For example, the protein may~be analyzed to determine an N-terminal sequence, or fragments of the protein can be produced by enzymatic or other specific digestion procedures and the sequence of the terminal W094/14g73 PCT~S93/12626 599 `~

amino acids of those fràgments determined. Such amino acid sequences, even-if only between five and six contiguous amino acids in length, will provide sufficient information to determine potential DNA
sequences of a gene enco~ing this protein.
If two or three such amino acid fragments are sequenced, a plurality of oligonucleotides can be synthesized using reported ~lOCedULe5~ and such oligonucleotides can be used to probe a genomic or CDNA
library from hookworm (or other source) to isolate the gene or fragments thereof enco~;ng the sequenced protein. Those in the art will r~cognize that these oligonucleotides can be designed using st~n~Ard parameters such that the oligonucleotide is ~hoe~n to encode the chosen amino acid sequence. For example, it is common to use a mixture of oligonucleotides as probes for any particular sequence of amino acids, with each oligonucleotide having the same nucleotide base sequence except at specific bases which are varied to take into account the redundancy of the codons that may code for any particular amino acid. It is of course desirable to select an amino acid sequence which is encoded by as few different oligonucleotides as possible. In addition, the various redundant coAon-e may be specifically selected to represent those codons that are most preferred in, for example, hookworm nucleic acid.
In addition, the isolated pure NIF protein can be used to obtain antibodies using known procedures. Such antibodies may include monoclonal or polyclonal antibodies and can be used to screen bacteriophage lambd-agtll expression libraries cont~;n;ng other source (e.a., hookworm) DNA. In this manner, any particular clone which includes nucleic acid encod~ng a NIF can be readily identified using stA~Ard procedures.
Genomic DNA libraries of a hookworm, for example, can be formed using standard procedures to isolate the W094/14973 PCT~S93112626 2~! ~259~g genomic DNA of the hookworm, fractionating that DNA
using either a random procedure, such as sonication, or a specific procedure such as restriction endonuclease digestion and ligation of those fragments into an appropriate vector, such as a bacteriophage lambda, plasmid or cosmid vector. Such a library can be screened for useful clones by nucleic acid hybridization using the oligonucleotide mixtures described above.
More preferably, however, a CDNA library can be constructed by isolation of total hookworm RNA, passage of that RNA~over an oligo-dT column to purify the poly(A)-containing RNA (i.e., messenger RNA), and reverse transcription of such RNA to produce DNA
- fragments representative of the RNA ( e., CDNA). These CDNA fragments can be inserted using stAn~rd ~Loced~res into any desired vector, for example, an expression vector such as a commercially available E. coli expression vector such as bacteriophage lambda-gtll (for expression in E. coli), or into a plasmid pcDNA-l which can be expressed in mammalian COS7 cells.
The biological activity of the protein expressed by individual clones of the plasmid expression library can be readily assayed using the neutrophil inhibitory activity assays described herein or other suitable assays. Alternatively, the antihoAies described above can be used to probe for immunoreactive protein expressed from clones in the bacteriophage expression libraries (e.g., lambda-gtll). Tt is particularly preferred to screen various libraries in sub-pools, for example of 999 clones at a time, to determine which of those sub-pools includes a positive clone. When a positive clone is isolated a grid of the 999 colonies can be formed on a 33 x 33 plate and ea~h of the 33 clones in each row and column in the plate assayed simultaneously (i.e., in 66 preparations) to identify the desired clone.

WO94/14973 PCT~S93/12626 S7~593 Once the desired clone is isolated, its structure is analyzed by stA~Ard procedures, for example, by DNA
sequencing to determine whether it encodes the whole of the desired protein. If it does not, that clone can be used to screen further CD~A or genomic libraries for full-length clones, or the DNA can be used to hybrid select RNA present in the hookworm, or other source, and more selective CDNA libraries formed from that RNA using procedures described above.
In another preferred aspect, the present invention is directed~to NIFs comprising an amino acid sequence which is encoded by a nucleic acid sequence which is sufficiently complementary to hybridize to CDNA probes derived from the amino acid sequence of a NIF.
Preferred are probes having at least about 12 nucleotides which are complementary to a portion of the sequence of Figure 8.
It should be apparent to those skilled in the art that the oligonucleotide primers can be used in the polymerase chain reaction (PCR) to generate complementary DNA probes. These probes can be used to isolate NIF from other sources or isoforms from a single source. Preferred are animal, fungal, bacterial or viral sources. In the PCR cloning method, single stranded DNA primers of 20-l00 nucleotides are derived from the sequence of NIF from Ancylostoma canium.
Preferred primers have the following characteristics:
limited degeneracy; adherence to codon usage preferences of the particular species from which the library is constructed and primers that target sequences which are conserved among the twelve Ancylostoma NIF isoforms.
Each PCR reaction utilizes two primers: a 5-primer that corresponds to the sense strand and a 34-primer that corresponds to the antisense strand of the NIF coding sequence. Especially preferred are the primers W094/14973 PCT~S93112626 2I~259g 5-CTCGAATTCT(GATC)GC(ATC)AT(ATC)(CT)T(GATC)GG(ATC)TGGGC-3' and 5'-CTCGAA~ l(TC)TCTGG(GA)AA(GA)CG(GA)TC(GA)AA-3'. The nucleotides within enclosing paren~hPc~c are redundant in that any one nucleotide may be used at the position enclosed by such parenth~s~s.
Single stranded CDNA template is generated using poly(A)+ or total RNA prepared from cells of the tissue or organism to be screened. RNA is primed with either random hexanucleotides or oligo d(T) and extended with reverse tran6criptase. This reaction product is amplified using an a~prG~Liate DNA polymerase (e.g., Taq polymerase), with a sense and anticenC~ primer, with an appropriate thermocycler.
A wide variety of polymerase chain reaction conditions may be employed, but initial experiments preferably involve relatively low stringency annealing and elongation steps. Preferred conditions are: cycles 1-3, denaturation at 94C for l minute, annealing at 37C for l minute and elongation at 72C for two minutes. The ramp time between annealing and elongation steps is extended to at least 2 minutes for these cycles; cycles 4-40, denaturation at 94C for l minute, annealing at 45C for l minute and elongation at 72C
for two minutes. In subsequent experiments, annealing temperature is increased until a single product results from amplification with each primer pair.
Amplification products from individual amplification reactions are used as hybridization probes to screen genomic DNA or CDNA libraries constructed from the tissue from which PCR was effected. DNA or CDNA
~ from any recombinant plaque or colony that hybridized to these amplification products is selected for further analyses.
NIF complementary DNAs isolated using the techniques described above are subjected to nucleotide W094/14973 PCT~S93/12626 sequence analysis using the procedure of dideoxy sequencing (Sanger et al, 1977, Proc. Natl. Acad. Sci J
USA 74:5463-5467).
NIF CDNA isolates cont~i ni ng open reading frames 5 (i.e., initiating with-a methionine and terminating with a TAA, TGA or TAG stop codon) are inserted into suitable vectors for protein expression in either bacterial, yeast, insect or mammalian cells. Expression systems comprise vectors designed to secrete recombinant protein 10 (i.e., fusion of CDNA isolate open reading frame with a known secre~ion signal sequence for that cell type) into the culture medium. Vectors lacking a secretion signal sequence are also used for expression. Either conditioned media or cell lysate, ~ep~n~ing on the 15 expression system used, is tested for inhibitory activity using one or more of the following criteria for neutrophil activation: release of hydroyen peroxide, release of superoxide anion, release of myeloperoxidase, release of elastase, homotypic ne~L~Gphil ayyre~ation~
20 adhesion to plastic surfaces, adhesion to vascular endothelial cells, chemotaxis, transmigration across a monolayer of endothelial cells and phagocytosis.
As discussed above and as described in Example lO, oligonucleotide primers derived from the peptide 25 sequences of NIF (isolated from the hookworm, Ancylostoma caninum) were used in conjunction with the polymerase chain reaction to amplify NIF CDNA sequences.
These NIF sequences were used in turn to probe a hookworm CDNA library. Ten full-length and six partial 30 clone isoforms of NIF were isolated in addition to the protypical NIF-lFL full-length clone. This example illustrates the utility of this technique for isolation of sequences that are structurally rela~éd to NIF.
Applicants note that by using tec~n;ques such as 35 those described above, as well as similar and equivalent techniques, DNA sequences which encode NIF from other WOg4/14973 PCT~S93/12626 animal, fungal, bacterial or viral source may be isolated and used to express recombinant NIF.
Should immunoreactive material be expressed from an expression library, the expression vectors described above, or derivatives thereof, can be used for expression of recombinant protein with biological activity. Such recombinant protein is useful in this invention.
Using one example of a NIF of the present invention, peptide fragments were produced and their amino acid 9equences determined. This experiment is described in Example 9. The amino acid sequences obtained for the proteolytic fragments are set forth in Figure 7.
An example of NIFs being cloned from a canine hookworm CDNA library as described in Examples 10 and 21. The nucleotide sequence for the CDNA of one of the isolated clones (clone lFL) is depicted in Figure 8.
Deduced partial amino acid sequences for other isolated NIF isoform clones are depicted in Figures 9 and 16.
By using the techn; ques described herein and other techniques in the art, NIFs may be isolated from any source, whether, animal, bacterial, fungal, viral or other source suspected of having a NIF. Such NIFs and nucleic acid sequences ~nco~;ng them may be isolated by methods such as probing a genomic or CDNA library from the source suspected of having a NIF using oligonucleotide probes sufficiently complementary to a nucleic acid sequence encoding a NIF such as those sequences depicted in Figure 8, and then isolating and expressing those nucleic acid sequences which hybridize ~ to the probes as described herein. Such probes have a sufficient number of nucleotides to des~ibe a unique sequence. Typically such probes will have at least about 12 nucleotides. One preferred group of probes include those of the sequences:

W094/14g73 PCT~S93112626 5'-CTCGAATTCT(GATC)GC(ATC)AT(ATC)-(CT)T(GATC)GG(ATC)TGGG
C-3' and 5'-CTCGAATTCTT(TC)TC-TGG(GA)AA(GA)CG(GA)TC(GA)AA-3'. The nucleotides within enclosing parentheses are ~ t in that any one nucleotide may be used at the position enclosed by such paren~hPcec.
Alternatively, NIF proteins and nucleic acids coding for such proteins may be isolated by probing a sample of nucleic acid from a source suspected of having a NIF with an oligonucleotide probe having at least about 12 nu~leotides which is complementary to a nucleic acid sequence known to encode a NIF, such as the sequence depicted in Figure 8 and isolating those nucleic acid sequences, such as a gene, which are sufficiently complementary to the oligonucleotide probe to hybridize thereto. The isolated nucleic acid sequence may then be cloned and expressed using art t~çhn;ques.

(D) Isolated Nucleic Acid Molecules.
In another aspect, the present invention is directed to isolated nucleic acid molecules comprising a nucleic acid sequence ~ncoAing the amino acid sequence of NIF. The DNA isolate may also include additional sequences which do not code for portions of the finished protein, such as introns, and/or sequences which code for intervening amino acid residues or peptides in addition to the above peptide se~nceC. Preferred isolated nucleic acid molecules are those which encode a NIF comprising at least one amino acid sequence selected from the group consisting of (a) Arg-X1-X2-Phe-Leu-X3-X4-His-Asn-Gly-Tyr-Arg-Ser-X5-Leu-Ala-Leu-Gly-His-X6-X7-Ile, wherein~X~ is Leu or Arg; X2 iS Gln, Lys or Arg; X3 is Ala or Arg; X4 iS Leu or Met; X5 iS Lys, Arg, Leu or Ile; X6 is Val or Ile; and X7 is Ser, Gly or Asn;

WO94tl4973 PCT~S93/12626 2152~99 (b) Ala-X8-Xg-Ala-Ser-X~O-Met-Arg-Xl~-Leu-Xl2-Tyr-Asp-Cys-XI3-Ala-Glu-Xl4-Ser-Ala-Tyr-Xl5-Ser-Ala, wherein X8 is His or Pro; X9 iS Thr, Arg or Ser; XlO is Arg or Lys;
Xl1 is Ile or Tyr; Xl2 is Asp, Lys or Glu; Xl3 is Asp or S Glu; Xl4 is Gly, Lys or Arg; and Xl5 is Glu, Met, Thr or Val;
(c) Ser-XI6-Phe-Ala-Asn-XI7-Ala-Trp-Asp-Xl8-Arg-Glu-Lys-XI9-Gly-Cys-Ala-Val-Val-X20-Cys, wherein Xl6 is Asn or Asp; X17 iS Val or Leu; X18 iS Ala or Thr; X19 iS Leu, Val or Phe; and X20 is Thr, Lys or Asn;
(d) H~s-Val-Val-Cys-His-X2l-X22-Pro-Lys, wherein X
is Tyr or Ile; X22 iS Gly or no residue;
(e) Ile~TYr~X23~X24~GlY~X2s~Pr~CYS~X26~X27~CYS~X2s~
X29-Tyr, wherein X23 iS Thr, Ser, Lys or Glu; X24 iS Thr, Val or Ile; X25 is Val, Lys or Thr; X26 is Arg, Ser or Asp; X27 iS Asn, Gly, Asp or Arg; X28 iS Asn, Ser or Thr;
and X29 iS Gly, Glu or Asp; and (f) Cys-X30-X3l-Asp-X32-Gly-Val-Cys-X33-Ile, wherein X30 is His, Ile or Asn; X3l is Ala, Pro or Asp; X32 is Glu, Val, Asp or Ile; and X33 iS Ile, Val or Phe. Especially preferred isolated nucleic acid molecules include those wherein its the co~;~g region has the nucleotide sequence and/or codes for a protein having the deduced amino acid sequence set forth in Figure 8.

2S (E) Ex~ression of NIF.
In another aspect, the present invention is directed to methods for making biologically active NIFs, wherein such NIFs are expressed intracellularly and are optionally secreted; expression vectors encoding NIF;
and host cells transformed with these expression vectors which express and, optionally, secrete NIF.
The CDNA encoding NIF may be insertëd into a replicable vector for expression, resulting in the synthesis of biologically active recombinant NIF. Many vectors are available for expression of heterologous W094114973 PCT~S93112626 ?,~5~99 44 proteins and selection of the a~L~iate vector will depend primarily on the desired properties of the host cell. Each of the available vectors contain various components specific to the host cell to be transformed.
The vector components or control elements generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, a promoter, an ~nhAncer element and a transcription termination sequence. Once the expression vector containing the inhibitor is constructed,- a suitable host cell is transfected or transformed with the expression vector, and recombinant NIF is purified either from the host cell itself or the host cell growth medium.
In general, the signal sequence may be a component of the vector, or it may be encoded by the NIF DNA that is inserted into the vector. If the native inhibitory factor is a secreted gene product (i.e., from the hookworm (or other source) cells), then the native pro-NIF from hookworm DNA may encode a signal sequence at the amino terminus of the polypeptide that is cleaved during post-translational processing of the polypeptide to form the mature NIF.
All vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacterial, yeast, insect and mammalian cells. The origin of replication from the plasmid PBR322 is suitable for most for most gram-negativ~ bacteria, the 2m plasmid origin is suitable for yeast, the baculovirus origin is suitable for some insect cells (e.q., Sf9 cells; ATCC# CRLl711) and various viral origins (e.q., WO94114973 PCT~S93112626 -SV40, adenovirus) are useful for cloning vectors in mammalian cells.
Expression vectors preferably contain a selection gene, also termed a selectable marker. This gene encodes a protein n~cecc~ry for the survival or growth of transformed host cells grown in selective culture medium. Host cells not transformed with the vector contA;n;ng the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g.~ ampicillin, neomycin or methotrexate, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
Expression vectors contain promoters that are recognized by the host organism. Promoters are untranslated sequences located ~L~eam (5') to the start codon of a structural gene (generally within about lO0 to lO00 base pairs) that ~G.1L.ol the transcription and translation of a particular nucleic acid sequence, such as hookworm NIF, to which they are operably linked.
A large number of promoters recogn;zed by a variety of potential host cells are well known. These promoters are operably linked to DNA encoding the NIF by inserting the latter into the vector in a way such that the 5' terminus of the NIF DNA is in close linear proximity to the promoter.
Transcription of a DNA ~nco~;ng the NIFs of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. (For ~ 30 example, see, Kriegler, M., l99l, Gene Transfer and Ex~ression, pages 4-18, W.H. Freeman, New York3.
Enhancers are cis-acting elements of DNA, usually about 10-300 base pairs in length, that act on~a promoter to increase its transcription. Enhancers are relatively orientation and position independent. Typically, one will use an enhancer from a eukaryotic cell virus for W094l14973 PCT~S93112626 -expression in mammalian cells. Examples include the SV40 enhancer, the cytomegalovirus early promoter enhancer and the adenovirus enhancers.
Expression vectors used in eukaryotic (i.e., non-bacterial) host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such se~enceC are commonly available from the 5' end and, occasionally from the 3' untranslated regions of eukaryotic or viral DNAs.
Preferred expression vectors of the present invention in~lude but are not limited to PHIL7SP-NlclO, PSG5/NIFlFLCRl, Pma5-NIl/3 PAN-NIF-lFL, pYAM7SP-hNIFl/~,G11-5, pYAM7SP-hNIFl/A,G11-5, pYAMSP-AcaNIF4, pYAMSP-AcaNIF6, pYAMSP-AcaNIF9, pYAMSP-AcaNIF24 and pAN-AceNIF3, the construction of which is described in the Examples.
Suitable host cells for the expression vectors described herein include bacterial, yeast, insect or mammalian cells. Preferred bacteria include E. coli strains, preferred yeast include SaccharomYces cerevisiae and Pichia pastoris, a preferred insect cell line is Sf9 (ATCC# CRL 1711) include preferred mammalian cell lines are COS-7 (ATCC# CRL 1651), CHO dhfr (ATCC#
CRL 9096), CHO-Kl (ATCC# CCL 61) and HeLa (ATCC# CCL 2).
These examples of host cells are illustrative rather than limiting. Preferably the host cell should secrete minimal amounts of proteolytic enzymes. Particularly suitable host cells for the expression of glycosylated NIF are derived from multicellular organisms. Such host cells are capable of complex post-translational processing and glycosylation of expressed proteins.
Host cells are transfected and preferably transformed with the above-described exp~éssion vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters and selecting transformants. Transfection refers to the WO94/14973 PCT~S93/12626 9,9 taking up of an expression vector by a host cell.
Numerous methods of transfection are known to the ordinarily skilled artisan, for example, calcium phosphate coprecipitation, spheroplasting transformation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.
Transformation means illL~od~cing DNA into an organism so that the DNA i8 replicable, either as an extrachromosomal element or chromosomal integration.
Depending o~ the host cell used, transformation is done using standard t hn;ques a~rop~iate to such cells (e.g., calcium chloride or ele~Lo~oration for bacterial cells; spheroplasting or ele~o~oration for yeast cells; calcium phosphate or ele~Lto~oration for insect and mammalian cells).
The recombinant hookworm NIF preferably is recovered from the culture medium as secreted polypeptide, although it may also be recovered from host cell lysates when expressed intracellularly without a signal or secretory sequence. The expressed hookworm NIF may be purified from culture medium or from cell lysates by a variety of separation te~hni~ues including, but not limited to, gel filtration, affinity and ion exchange chromatography, hydroxyapatite chromatography, C4 reverse-phase HPLC and preparative isoelectric chromatography.

(F) Mutant NIFs.
In another aspect, the present invention is directed to mutant NIFs comprising the amino acid sequence shown in Figure 8, wherein one or more of asparagine residues at positions lO, 18,~87, llO, 130, 197 or 223 is replaced by an glutamine residue.
Amino acid sequence variants of NIF may be prepared by introducing nucleotide changes into the DNA which ?,~5?J599 ; - -encodes NIF, isolated as described above. Such variants include substitutions of residues within the amino acid sequence of the NIF. Any combination of substitutions can be made to arrive at the final construct, provided 5 that the final construct roCcecces certain desired characteristics. The desired characteristics include, but are not limited to, an increased potency over the wild-type NIF and alteration of the amount of glycosylation of NIFs. Once variant NIF DNAs have been 10 constructed, variant recombinant forms of NIF may be synthesized-utilizing expression systems as described above.
one possible method for preparing variants of NIF
is mutagenesis with base-specific chemical mutagens as 15 described in detail by Pine and Huang (1987, Methods Enzymol. 154, 415-430). Another approach is site-directed mutagenesis, for example, as described in StAncs~c et al. (1989), Nucl. Acids Res., 17:
4441-4454. For example, Example 25 describes the 20 substitution of certain asparagine residues in the amino acid sequence of a NIF referred to as lFL (see Figure 8) by site-specific mutagenesis.
The asparagine residues at positions 10, 18, 87, 110, 130, 197 and 223 of the amino acid sequence of NIF
25 isoform, lFL, are believed to be sites associated with the potential N-linked glycosylation of this isoform.
Using the stepwise site-directed procedure of Stanssens et al. and the five oligonucleotides described in Example 25, asparagine residues at positions 10, 18, 87, 30 110 and 130 of lFL were replaced by glutamine residues to yield a mutant NIF. Subsequently, the cDNA encoding this mutant was further mutagenized by the same procedure using other oligonucleotides d~scribed to produce a mutant NIF, wherein the asparagine residues at 35 positions 197 and 223 of the amino acid sequence of lFL
were also replaced with glutamine residues. In either W094/14973 PCT~S93/12626 case, the expressed mutant NIFs were found to have neutrophil inhibitory activity.

2. Pe~tide Fraaments.
In another aspect, the present invention is directed to peptide fragments having neutrophil inhibitory activity which are prepared by proteolytic or chemical methods starting with the chromatographically pure NIF of the present invention.
Active peptide fragments, with or without sugar moieties, ma~ be generated by using enzymatic or chemical techniques. Proteolytic cleavage can be accomplished by digestion of the inhibitor withS one or more of the following enzymes: chymotrypsin, trypsin, leucine aminopeptidase, endoproteinase Glu-C, endoproteinase Lys-C, Pn~oprot~in~se Arg-C, or endoproteinase Asp-N (Carrey, E.A., 1989 Protein Structure A Practical Approach, pp. 117-143, T.E.
Creighton, ed. IRL Press, New York). Chemical digestion of the inhibitor may be accomplished by cyanogen bromide, hydroxylamine, or 2-nitro-5-thiocyanobenzoate cleavage (Carrey, E.A., 1989, ibid.). Sugar moieties can be removed from either the peptide fragments or intact neutrophil inhibitory protein enzymatically with one or more of the following enzymes: glycopeptidase F, endoglycosidase H, endoglycosidase F, or endoglycosidase D as described by Keesey (Keesey, J., 1987 Biochemica Information, pp. 147-165, J. Keesey, ed., Boehringer Mannheim Biochemicals, Indianapolis). Alternatively, glycosylation of the intact inhibitor may be suppressed by expression of the protein in bacterial cells or by ~ the inclusion of inhibitors of glycosylation in the eukaryotic cell culture growth medium. I~hibitors of glycosylation and their uses are described in the art (e.g., Keesey, J. 1987 Biochemica Information, pp.
135-141, J. Keesey, ed., Boehringer Mannheim W094J14973 PCT~S53tl2626 ~ 5~599 ` 50 Biochemicals, Indianapolis). Separation of active fragments from inactive fragments may be accomplished by conventional, low, medium, or high pressure chromatographic techn;ques known in the art.

3. Antibodies.
In another aspect, the present invention is directed to polyclonal and monoclonal an~; ho~; es which have the ability to bind to NIFs.
To prepare antibodies to NIF, any one of a number of conventio~al ter~n;ques which are known in the art can be employed. In one such t~ch~;que, polyclonal ant;hoAies are synthesized by injecting an animal (for example a rabbit) with one or more NIF of the present invention. After injection, the animal produces antibodies to these NIFs. When the antibody concentration (or titer) reaches a sufficient level, antibody-containing blood is then drawn from the animal, antiserum is prepared from the blood, and the compound-specific antibody is isolated from other antibodies in the serum by any one of a number of separation techn;ques (for example, affinity chromatography).
Nonoclonal antibodies may be prepared using the techniques of Kohler and Milstein, Nature 256, 495-497 (1975) as well as other conventional techniques known to those skilled in the art. (See, e.a., Harlow and Lane, Antibodies. A LaboratorY Manual (Cold Spring Harbor Laboratory, 1988) the disclosures of which is incorporated herein by reference). Preferred monoclonal antibodies include those directed to the NIF isoform, lFL, whose amino acid sequence is depicted in Figure 8 and which are immunoglobulins of the Ig~ class.
Especially preferred monoclonal an~;ho~;es are those which bind to the same epitope on this NIF as is bound by the monoclonal antibody, 3D2. The monoclonal W094/14g73 PCT~S93/12626 -antibody referred to as 3D2 is a preferred embodiment.
The preparation of 3D2 is described in Example 26.
In another aspect, the present invention is directed to hybridomas which produce such monoclonal antibodies. These hybridomas are produced by conventional tech~; ques such as those described by Harlow and Lane, Id., the disclosures of which is incorporated herein by reference. The preparation of a preferred hybridoma is described in Example 26.
In another aspect, the present invention is directed to~methods of affinity purification of NIF from various sources and impure compositions of NIF derived from such sources using an antibody to NIF. Preferred method of isolating NIF would comprise the step of contacting a sample thought to contain a NIF with a monoclonal antibody which is capable of binAi~g to said NIF. Preferred as the monoclonal antibody is either the monoclonal antibody, 3D2, or a monoclonal antibody binding to the same epitope on said NIF as is bound by the monoclonal antibody, 3D2. According to preferred methods of affinity purification the monoclonal antibody is covalently attached to a chromatographic resin.
Especially preferred chromatographic resins include Emphaze~ BiG-u~ul~ Medium (3M Corp.). Examples 27 and 28 describe preparation of a chromatographic resin coupled with the monoclonal antibody, 3D2, and its use to purify NIF from compositions comprising NIF.
In another aspect, the present invention is directed to immunoassays using the antibodies against ~ 30 NIF. Depending on the particular use for the immunoassay, an immunoassay format is selected. Some suitable immunoassays are described by Harlow and Lane, Id. (See especially pages 553 to 612)~ the disclosures of which are incorporated herein by reference.
Immunoassays utilizing the solid phase method or liquid phase method are well known to one skilled in the WO94/14973 PCT~S93/12626 5g9 ~` 52 art of immunoassays. For example, monoclonal antibodies may be used to assay for drugs, hormones and proteins with such assays being in a solid phase or liquid phase format. The preferred methods of the present invention are solid phase assays.
The preferred methods of detecting NIF in a sample comprise contacting a sample thought to contain a NIF
with a monoclonal antibody which is capable of binding to such NIF. According to a preferred emhoA;ment~ the monoclonal antibody is immobilized onto a plastic surface su~h polystyrene, pol~LGpylene, polyethylene, nylon and the like. The plastic surface may be configured in the shape of test tube, microspheres, macroscopic beads, microtiter plates and the like. In this emhoAiment, monoclonal antibodies may be attached to the plastic surface by either covalent coupling or by passive absorption, preferably by passive absorption.
Preferred as monoclonal ant;hoAPC are those which bind to the same epitope on a NIF as is bound by the monoclonal antibody, 3D2, or the monoclonal antibody, 3D2.
The monoclonal antibody may be contacted simultaneously with sample and a NIF which has been covalently linked to a detectable label. Alternatively, the monoclonal antibody may first be contacted with the sample, then with a NIF which has been covalently linked to a detectable label.
Preferred detectable labels are enzymes, fluorescent compounds or radioisotopes. Especially, preferred detectable labels are enzymes such as alkaline phosphatase, ~-galactosidase or horseradish peroxidase, or radioisotopes such as iodine-125. The manner of covalently linking such enzymes and r~ioisotopes to monoclonal antibodies is well known to one skilled in the art of diagnostic assays.

WO94/14973 PCT~S93/12626 2l~2~y 4. Methods of Detectinq NIF Mimics and NIF
Antaqonists .
In another aspect, the present invention is directed to a method of detecting in a sample the presence of a NIF mimic which competes with NIF for binding to CDllb/CD18 ~ecepLor or the I-domain portion of the CDllb/CD18 receptor., In another aspect, the present invention is directed to a NIF antagonist which prevents NIF from binding to the CDllb/CD18 receptor or the I-domain portion of the CDllb/CD18 receptor. The methods co~prise contacting said sample with CDllb/CD18 receptor or the I-domain portion of the CDllb/CD18 receptor. NIF mimics and NIF antagonists include but are not limited to small molecules, peptides, peptide analogs or proteins.
In preferred emhoA;ments, NIF mimics or antagonists to be tested are preincubated in solution with neutrophils, or immobilized CDllb/CD18 receptor or a recombinant peptide comprising the I-domain portion of the CDllb/CD18 receptor, and the preincubated solution is then brought into contact with labeled NIF. The effect of test compound on the binding of NIF to neutrophils, immobilized CDllb/CD18 receptor or recombinant peptide comprising the I-domain portion of the CDllb/CD18 receptor is then determined.
In one preferred emhoAiment~ the assay method uses neutrophils which are free in solution. Here, NIF which has been linked to a detectable label, neutrophils and a sample thought to contain a NIF mimic or NIF antagonist are co-incubated in solution for a sufficient time to allow binding to occur. Unbound~ labeled NIF is removed from bound NIF by methods such as centrifugation, filtration or other suitable methods and bound NIF is determined by means of the detectable label.
In another preferred embodiment, neutrophils are immobilized on a plastic surface by passive absorption W094/14973 PCT~S93/12626 a99 or chemical fixation such as by glutaraldehyde or similar chemicals. Labeled NIF is co-~ ~c"hAted with the immobilized neutrophils and a sample thought to contain a NIF mimic or NIF antagonist. Unbound labeled NIF is removed by washing and bound labeled NIF is then determined by means of the detectable label.
In an especially preferred embodiment, CDllb/CDl8 receptors from a detergent extract of human leukocytes are captured by anti-CDllb/CDl8 monoclonal antibody which is immobilized to a plastic surface. A NIF which is linked ~o a detectable label or a NIF which can be subsequently linked to a detectable label, and sample thought to contain a NIF mimic or NIF antagonist are co-incubated with the immobilized CDllb/CDl8 Lec-pLor.
After the binding has G~ Led, unbound NIF is removed by washing. Detectable label is then added which links to NIF rendering it detectable (if the NIF was not originally linked to such label). Bound NIF is then determined by means of the detectable label.
Anti-CDllb/CDl8 antibody may be coupled to a plastic surface by covalent coupling or passive absorption, though passive absorption is preferred.
Preferred plastic surfaces are poly~yLene, pol~L~ylene, polyethylene, nylon and the like, though polystyrene is preferred. The plastic surface may be configured in the shape of test tube, microspheres, macroscopic beads, microtiter plates and the like. A
preferred anti-CDllb/CDl8 antibody is the monoclonal antibody referred as LM2.
3 0 NIF is linked to a detectable label or is capable of being linked to such label during a step of an assay method of the present invention. NIF is linked to a detectable label by covalent coupling~using homobifunctional crosslinking reagents such as 3 5 glutaraldehyde, disuccinimidyl suberate, dimethyl suberimidate and the like. NIF is made capable of being Wo ~/14973 PCT~S93/12626 21~2599 linked to a detectable label during a step of an assay method of the present invention by first linking biotin to NIF and avidin to the detectable label. Preferred detectable labels include enzymes, fluorophores or radioisotopes. Especially preferred detectable labels include alkaline phosphatase, ~-galactosidase, horseradish peroxidase, or iodine-125.
In another especially preferred embodient, a recombinant peptide comprising the I-domain of the lo CDllb/CDl8 receptor which is complexed to a monoclonal antibody that reco7nizes a portin of this peptide NIF
which is linked to a detectable label or a NIF which can be subsequently linked to a detectable label, and sample thought to contain a NIF mimic or NIF antagonist are co-incubated. After the b;n~ng has or-~LLed, a protein A-Sepharose is added to separate bound from unbound NIF.
Setectable label is then added which links to NIF
rendering it detectable (if the NIF was not originally linked to such label). Bound NIF is then detrmined by means of the detectable label.
A NIF mimic or NIF antagonist may be assayed for neutrophil inhibitory activity. NeuLLGphil inhibiting activity may be demonstrated by an assay such as those assays which determine adhesion of ne~L.o~hils to vascular endothelial cells, relèase of hydLG~en peroxide from neutrophils, homotypic neuLl~hil aggregation and adhesion of neuLLG~hils to plastic surfaces. NIF mimics are characterized as having an IC50 for inhibiting neutrophil activity of about 500 nM or less, though an ICso is about 100 nM or less is preferred. NIF
antagonists are characterized having such an IC50 of about l,OoO nM to about lmM, although an IC50 of about 5,000 nM to about 10 mM is preferred. An IC50 is that concentration of a NIF mimic or NIF antagonist giving 50% inhibition of the measured activity (see Example 1).

WO ~/14973 PCT~S93/12626 599 `-Optionally a NIF mimic or NIF antagonist may be assayed for eosinophil inhibitory activity. Eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells. If assayed, NIF mimics are characterized as having an IC50 for inhibiting eosinophil activity of about 500 nM or less, though a IC50 is about 100 nM or less is preferred. If assayed, NIF
antagonists are characterized having such an IC50 of about 1,000 nM to about 1 mM, though an IC50 of about 5,000 nM ~o about 10 mM is preferred. An IC50 is that concentration of a NIF mimic or NIF antagonist giving 50~ inhibition of the measured activity.
In another aspect, the present invention is directed to NIF mimics and NIF antagonists discovered by the above-disclosed methods of detection of this section.

5. Pharmaceutical Formulations and Methods of Treatment.
In another aspect, the present invention is directed to pharmaceutical compositions comprising NIF.
These pharmaceutical compositions may be formulated and used as tablets, capsules or elixirs for oral administration; ~u~yo~itories for rectal administration;
sterile solutions, suspensions for injectable administration; and the like. The dose and method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
Generally, an amount between 0.01 mg/kg to 100 mg/kg body weight/day is administered dependént upon the potency of the composition used.
Preferred embodiments encompass pharmaceutical compositions prepared for storage and subsequent W094/14973 PCT~S93/12626 '~, administration which comprise a therapeutically effective amount of NIF or an enriched composition of NIF, as described herein in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publ;ehing Co.
(A.R. Gennaro edit. 1985). Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspen~ing agents may be used.
In another aspect, the present invention is directed to methods of preventing in a mammal an inflammatory condition characterized by abnormal neutrophil activation or abnormal eosinophil activation comprising administering to said mammal a therapeutically effective amount of a NIF or their pharmaceutical compositions. In practicing the preferred methods, NIFs or their pharmaceutical compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These compositions can be utilized in vlvo, ordinarily in a mammal, preferably in a human, or in vitro.
In employing NIFs or their pharmaceutical compositions in vivo, the compositions can be administered to the mammal in a variety of ways, - 30 including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms.
As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the mammalian species treated, the particular WO94114973 PCT~S93/12626 2~5g9 58 composition employed, and the specific use for which these compositions are employed. The determination of effective dosage levels, that is the dosage levels neC~cs~ry to achieve the desired result, will be within the ambit of one skilled in the art. Typically, applications of compositions are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved.
The dosage for a NIF or its pharmaceutical compositions can range broadly ~p~n~;~g upon the desired ef~ects and the therapeutic indication.
Typically, suitable dosages will be between about O.ol mg and lO0 mg/kg, preferably between about O.Ol and lO
mg/kg, body weight. Administration is preferably parenteral, such as intravenous on a daily or as-needed basis.
Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydLochloride or the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic al~Y~ ry subst~nce-c, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (e.g., liposomes) may be utilized.

6. UtilitY and AD~lications As applicant has noted abov~e, the present invention is directed to inhibitors of the leukocytes, and in a preferred aspect, to those leukocytes which have or express CDllb/CDl8 receptors. Leukocytes having or expressing the CDllb/CDl8 integrin receptor are well known and include the monocytes, macrophages, WO94/14973 PCT~S93112626 ` .~15~599 granulocytes, large granular lymphocytes (NK cells), and immature and CD5+ B cells (Xishimoto, T.K., Larson, R.S., Corbi, A.L., Dustin, M.L., Staunton, D.E., and Spriger, T.A. (1989) Adv. in Immunol. 46,149-182).
CDllb/CD18 has been implicated in a variety of leukocyte functions including adhesion of ne~Llu~hils to endothelial cells (Prieto, J., Beatty, P.G., Clark, E.A., and Patarroyo, M. (1988) Immunology 63, 631-637;
Wallis, W.J., Hickstein, D.D., Schwartz, B.R., June, C.H., Ochs, H.D., Beatty, P.G., Klebanoff, S.J., and Harlan, J.M.~ (1986) Blood 67, 1007-1013; Smith, C.W., Marlin, S.D., Rothlein, R., Toman, C., and Anderson, D.C. (1989) J. Clin. Invest. 83, 2008-2017) and release of hydrogen peroxide from neutrophils (Shappell, S.B., Toman, C., Anderson, D.C., Taylor, A.A., Entman, M.L.
and Smith, C.W. (1990) J. Immunol. 144, 2702-2711; Von Asmuth, E.J.U., Van Der T ;n~n~ C.J., Leeuwenberg, J.F.M., and Buurman, W.A. (1991) J. Immunol. 147,3869-387S). This integrin may play a roll in neuL.Gphil and monocye phagocytosis of opsonized (ie C3bi-coated) targets (Beller, D.I., Springer, T.A., and Schreiber, R.D. (1982) J.Exp. Med. 156,1000-1009). It has also been reported that CDllb/CD18 contributes to elevated natural killer activity against C3bi-coated target cells (Ramos, O.F., Kai, C., Yefenof, E., and Klein, E. (1988) J. Immunol. 140,1239-1243).
Also noted previously, the NIFs of the present invention have potent neutrophil inhibitory activity and, thus, may be used as an inhibitors of neutrophil activity, including neutrophil activation in vitro, as well as for preventing or treating in a mammal inflammatory conditions characterized by abnormal neutrophil activation.
Thus, NIF will be useful in the treatment of inflammation in which the abnormal activation of neutrophils plays a significant role. While applicants W094/14973 PCT~S93112626 do not wish to be bound to any theory or mode of activity, it is believed that this compound will interfere with the inflammatory response which is set into action by neutrophil-endothelial cell interactions.
Thus, where adhesion of neuLl~hils to the endothelium is prevented, the neutrophils will be unable to transmigrate to tissue t~ elicit a proinflammatory response with consequent tissue damage. Inhibition of neutrophil-neutrophil adhesion and/or aggregation by these NIFs should also prevent microvascular occlusion.
Thus, these NIFs will be useful in treating a variety of clinical disorders, including shock, stroke, acute and chronic allograft rejection, vasculitis, autoimmune diabetes, rheumatoid arthritis, inflammatory skin diseases, inflammatory bowel ~iF~ce~ adult respiratory distress syndrome (ARDS), ischemia-reperfusion injury following myocardial infarction, in which neutrophil infiltration and activation has been implicated and acute inflammation caused by bacterial infection, such as sepsis or bacterial meningitis.
The ability of NIF to inhibit neutrophil activity makes it useful in inhibiting the physiological processes of inflammation, ischemia, and other neutrophil mediated tissue damage. The specific activities of NIFs in carrying out these related functions makes it particularly useful as therapeutic and/or diagnostic agents.
Antibodies, both monoclonal and polyclonal, directed to NIF are useful for diagnostic purposes and for the identification of concentration levels of the subject peptides in various biological fluids.
Immunoassay utilizing these ant;ho~;es may be used as a diagnostic test, such as to detect inf~ction of a mammalian host by a parasitic worm or to detect NIF from a parasitic worm in a tissue of the mammalian host.
Also such immunoassays may be used in the detection and WO ~/14973 PCT~S93112626 ~ 2I52599 isolation of NIF from tissue homogenates, cloned cells and the like.
In another aspect of the present invention, NIFs can be used in a test method to screen other compounds to detect NIF mimics or to detect NIF antagonists for their ability to affect NIF b;n~;n~ to the CDllb/CDl8 receptor.
In yet another aspect of the present invention, NIF
with suitable adjuvants can be used as a vaccine against parasitic worm infections in mammals. Immunization with NIF vaccine~may be used in both the prophylaxis and therapy of parasitic infections.~ NIF fragments and synthetic polypeptides having the amino acid sequence of NIF may also be used as vaccines. Disease conditions caused by parasitic worms may be treated by administering to an animal infested with these parasites substances which antagonize NIF (such as NIF
antagonists). Compounds may be screened for their anti-NIF effect according to the scr~en;ng method described herein above. Examples of such antihelminic agents include ant;hoA;es to NIF, both naturally occurring ant;ho~;es isolated from serum and polyclonal and monoclonal ant; ho~; es described above. Chemically synthesized compounds which act as inhibitors of NIF
also are suitable antihelminic agents.
To assist in understAn~;ng the present invention, the following examples are included which describe the results of a series of experiments. The following examples relating to this invention should not, of course, be construed as specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are oonsidered to fall within the scope of the invention as described herein and hereinafter claimed.

W094/14973 PCT~S93112626 g9 ` 62 Examples Example l Assays of Neutrophil InhibitorY Activity The Neutrophil Inhibitory Factor of the present invention demonstrated activity in inhibiting neu~Lu~hil function as measured by neutrophil-HWEC and neutrophil-plastic adhesion assays, homotypic neutrophil ayyLeyation assay and 1.ydLoyen peroxide release assay.
This inhibitory factor was isolated from hookworm tissue lysates as an enriched composition by a variety of methods in~luding gel filtration chromatography, chromatography on hydroxyapatite and concanavalin A
sepharose, C4 r e~el~e-phase HPLC, Mono-Q ion exchange chromatography and preparative isoelectric focusing. The isolated factor AppeArs to inhibit neu~LGphil adhesion to endothelial cell monolayers by inhibiting neutrophil activation.

(A) Cells and Reaaents Primary human umbilical vein endothelial cells (H WEC), obtAi~e~ from Clonetics (San Diego, CA), were - maintained in EGM-W medium (Clonetics) with 15% fetal bovine serum (FBS), in a 5% CO2 atmosphere. HUVEC were passaged twice and used to seed fibronectin-coated 96 well microtiter plates (Collaborative ReceArch, Bedford, MA) for adhesion assays.
The protease inhibitors E64, pepstatin A, chymostatin and APMSF were obtained from Calbiochem (La Jolla, CA).
Neutrophils were isolated using Mono-Poly resolving medium (ICN Biomedicals, Costa Mesa, CA) from either heparinized or citrated human blood following the instructions of the manufacturer. Ne~Lo~hils were resuspended in HSA buffer (RPMIl640 with lO mM HEPES pH

7.4, l.2 mM CaCl, l.0 mM MgCl, 1% human serum albumin) WO ~tl4973 PCT~S93/12626 -at a concentration of 6.6x106 cells/ml and used within one hour after isolation.
Neutrophils were fluorescently labelled by the ; following procedure. The cells were washed once in Hank's balanced salt solution (HBSS) and resuspended at lx107 cells/ml in HBSS containing 20 mg/ml calcein (Molecular Probes; Eugene, OR). The calcein was initially solubilized in 50 ml dry dimethylsulfoxide prior to its addition to the HBSS. Cells were incubated at 37C with occasional mixing by inversion. After 45 minutes incùbation the cells were chilled on ice for 5 minutes and then washed twice with ice-cold HSA buffer.
Labelled neutrophils were resuspended in HSA buffer at 1.3x107 cells/ml for use in adhesion assays.

(1) Protein concentration.
The molar protein concentration of purified NIF
isoforms and mutants thereof was determined spe~L~ophotometrically at 278 nm thereby using calculated extinction coefficients. The calculation is based on absorbance values of 5600 cm~~.mol-~ for tryptophan and 1420 cm~~.mol-~for tyrosine residues.

NIF-lFL AcaNIF4 AcaNIF6 AcaNIF24 Trp-residues 2 3 2 2 Tyr-residues 13 20 17 17 ~M 29,660 45,200 35,320 40,920 (2) Pre~aration of 3D2-HRP conjugate.
Two hundred fifty microliters glutaraldehyde (25%
solution) was added to 1 ml of an 8 mg/ml solution of horseradish peroxidase (Sigma P8375; in phosphate buffered saline (PBS)). After 2 hours at room temperature the mixture was transferred to a dialysis bag (MW cut-off 12kD) and dialysed against 1 liter PBS
for 5 hours. Buffer was refreshed three times. After WO ~/14973 PCT~S93/12626 ?,~S?~599 64 transfer of the solution to a test tube an equal volume of 3D2 purified MAb (see Example 26; dissolved in PBS at a concentration of 2mg/ml) was added and further incubated overnight at room temperature. The reaction was stopped by adding glycine to a final concentration of 50 mM. Bovine serum albumin (BSA; Calbiochem) was added to a final concentration of 0.25% (w/v) and the solution was aliquoted an`d stored at -20-C.

(3) Pre~aration of biotinylated Pichia NIF-lFL.
Purified recombinant NIF (NIF-lFL) from Pichia was dialyzed against 100 mM acetate buffer pH 5.5 at a concentration of lmg/ml. An equal volume of cold 20 mM
sodium-metaperiodate in 100 mM acetate buffer pH 5.5 was added. The oxidation reaction was allowed to proceed for 20 minutes in the dark on ice. The reaction was stopped by adding glycerol to reach a final concentration of 15 mM. The sample was desalted by ultrafiltration on Centricon 30. Biotin-LC hydrazide (Pierce) dissolved in dimethylsulfoxide was added to reach a final concentration of 5 mM and was then further incubated for 2 hrs at room temperature. The biotinylated sample was subsequently dialyzed against PBS using a Spectrapor membrane with a cut-off of 12,000.

(B) Neutrophil-HUVEC Adhesion AssaYs Calcein-labelled neuL u~hils (175 ml at 1.32x107 cells/ml) were preincubated for 10 minutes at room temperature with 175 ml of test fraction (diluted in HSA
buffer) in the presence of 160 nM phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO). PMA is solubilized in dimethylsulfoxide at a stock concentration of 1.6 mM. A 96 well p~te was used for this assay. One hundred microliters of this suspension was then aliquoted into each of three replicate wells that contained H W EC monolayers. Neutrophils were W O 94/14973 PCTrUS93/12626 `_ 2IS2599 incubated with the ~UVEC monolayer for 30 minutes at 37C. To remove non-adherent cells, wells were first filled with 250 ml HSA buffer, sealed with parafilm and then centrifuged inverted for 3 minutes at 75 X g.
Inverted plates were then placed on a rocking platform shaker for 5 minutes, after which contents were decanted off and wells were washed twice with 100 ml HSA buffer.
Adherent neuLLu~hils were lysed in 100 ml 0.1% (v/v) Triton X-100 (in 50 mM Tris HCl pH 7.4), and agitated for 10 minutes on a plate ~hAker. Twenty five microliter~ of the ne~LLo~hil/endothelial cell lysate was transferred to a 96 well microtiter plate that contained 100 ml of 50 mM Tris pH 7.4, and the wells were read at 530 nm (485 nm excitation) on a Cytofluor fluorometric plate reader (Millipore; Bedford, MA).
The hydroxyapatite pool preparation of hookworm Neutrophil Inhibitory Factor (see Example l(D)) inhibited neutrophil adhesion to H WEC monolayers with an IC50 of about 10 nM.

(C) Neutrophil-Plastic Adhesion Assay (1) Protocol #1.
This assay was used in Examples 1 through 19, where it was applicable.
Neutrophils (20 ml at 6.6X106 cells/ml) were incubated with 5 ml PMA (0.8 mM) for 5 minutes at room temperature in a 0.5 ml pol~G~ylene test tube. Twenty microliters of test fraction, diluted in HSA buffer, was added and the suspension was mixed gently. Aliquots of 10 ml of this suspension were added in triplicate to microtiter wells of 60-well HCA (Terasaki) plates (Nunc, - Naperville, IL). Neutrophils were incubated 5 minutes at 37C and non-adherent cells were re~oved by submerging the plate 6 times in HBSS.
Adherent neutrophils were quantitated by counting under an inverted light microscope. Binding was W094/14g73 PCT~S93/12626 ~,~52S99 quantitated visually. ^PMA-activated neutrophils spread and adhere tightly to polystyrene plastic.
Non-activated neuL~ophils (i.e., in the absence of PMA) remain round and translucent and do not adhere tightly to plastic. Adherent neuLlo~hils were larger, rhomboid in shape and more opaque, with a gr~nlllAr appearance.
In the absence of Neutrophil Inhibitory Factor, greater than 80% of PMA-activated neuLLo~hils rapidly and irreversibly bound plastic, underwent shape change and were not removed by the gentle wash procedule.
Moreover, ~actions cont~;ning the Ancylostoma Neutrophil Inhibitory Factor exhibited a profound inhibitory effect on plastic binding by activated neuL~u~hils.
The hyd~oxrapatite pool preparation of hookworm Ne~LL~hil Inhibitory Factor (see Example l(D)) inhibited neuL~v~l.il adhesion to plastic in this assay with an IC50 of about 10 nM.

(2) Protocol #2.
This assay was used in Examples 20 through 29, where it was applicable.
Neutrophils (60 ~1 at 8xlO6cells/ml in HSA buffer) were incubated with 15 ~1 PMA (2.4 mM) and 5 ~1 CaC12 0.05 M. After gently mixing 80 ~1 of the stimulated cell suspension were added to 96-well high binding polystyrene microtiter plates (Costar) cont~in;ng 20 ~1 of the test sample diluted in HSA buffer. After 45 minutes incubation at 37-C, non-adherent cells were removed by submerging the plates 4 times in PBS.
Adherent cells were loaded with dye by adding 100~1 of 0.5% (w/v) Crystal violet indicator (CAS 548-62-9) solution. After 10 min at room temperature plates were rinsed by submerging 4 times in PBS. Lysis of stained adherent cells was done by adding 100~1 of 1% (v/v) Triton X-100 solution. Absorbance was determined at WO94tl4973 PCT~S93/12626 21725g9 605nm with a Thermomax plate reader to quantitate adherent ne~ hils.

(D) HomotYpic Neutro~hil A~e~ation Neutrophil aggregation was performed at 37C in a Scienco dual channel aggregometer (Morrison, CO).
Neutrophils (190 ml at 6.6Xl06 cells) were preincubated with 200 ml test fraction (diluted in HSA Buffer) in a glass cuvette (Scienco) for 2 minutes at room temperature. Ten microliters of PMA were added to initiate ~yreyation (80 nM final). The inhibition of neutrophil ayyley~tion was measured at the maximum aggregation response 5 minutes after the addition of PMA.
The hydroxyapatite pool preparation of Neutrophil Inhibitory Factor (see Example l(D)) inhibited neuLLGphil adhesion with an IC~ of about 10 nM.

(E) Hyd~G~en Peroxide Release Assay Neutrophils (6.6X106 cells/ml) were ;ncllh~ted with test fractions in Release Assay Buffer (HBSS with 25 mM
glucose, 10% FBS, 200 mg/ml phenol red, 32 mg/ml horseradish peroxidase) for 5 minutes at 37C.
Incubation veCc~l~ consisted of 1.5 ml plastic test tubes that were precoated with HBSS containing 50% FBS
at 37C for 60 minutes; coated tubes were washed twice with 0.15 M NaCl before use. FMlP (Sigma; St. Louis, MO) at a final concentration of 250 mM was added and the neutrophil/test compound suspension was incubated at 37C for 60 minutes. Cells were pelleted by centrifugation at 8000 X g for 3 minutes and 200 ml of supernatant was transferred to a 96 well microtiter plate. Ten microliters of 1 N NaOH was added to each well and absorbance was read at 610 nm with a Molecular Devices ThermoMax plate reader. Hydrogen peroxide WO ~/14973 PCT~S93/12626 2~5~99 ~3, concentrations were determined by using a stAn~rd curve. Data points were done in duplicate.
The hydroxyapatite pool preparation of hookworm Neutrophil Inhibitory Factor inhibited hydrogen peroxide release from neutrophils with an IC50 of about 10 nM.

(F) CDllb/CD18 Bindina AssaYs Microtiter polystyrene plates (Costar - high binding; 96 well) were coated with the CDllb/CD18 binding, non-neutralizing mouse monoclonal antibody LM2 (50 ~l of th~ purified LM2 MAb at a concentration of 10 ~g/ml in 0.1 M NaHCO3;pH 9.5; LM2 ATCC hybridoma #HB204) by overnight incubation at 4 C. After removal of the antibody, the wells were blocked with 200 ~l PBS
contAini~g 1% (w/v) Skim-milk (Difco Laboratories) at room temperature. After 2 hours the blocking solution was removed and wells were washed 3 times with 200 ~l of PBS.
The immobilized LM2 monoclonal antibody was used to immuno-capture the detergent solubilized CDllb/CD18 receptor as follows. Ne~LLo~hils were isolated using Polymorphprep~ (Nycomed)-from either citrated whole blood or from buffy-coat following the instructions of the manufacturer. The neuLLo~hil pellet from a 50 ml buffy coat was resuspended in 40 ml RPMI and phorbol myristate acetate (PMA) to a final concentration of 0.8 ~M was added. The suspension was gently rotated at room temperature for 20 minutes. The cells were spun down and resuspended in 5 ml 0.02 M Tris-HCl, 0.15 M NaCl, o.oo1 M MgCl2, O.OQl M CaCl2. Cells were lysed by adding 5 ml buffer containing 2% Triton X-100 (Bio-Rad Laboratories), 0.02 M Tris pH 7.5, 0.15 M NaCl, 0.001 M
MgCl2, O.001 M CaCl2 and 0.02% thimerosa~ (Sigma T-5125).
PMSF (Sigma) and iodoacetamide (Merck-Schuchardt) were added to final concentrations of 1 mM. After mixing, the suspension was stored on ice for 1 hour and vortexed 2152~gg periodically. After centrifugation at 30,000 g, the supernatant was diluted twofold with 0.02 M Tris-HCl, 0.15 M NaCl, 0.001 M NgCl2, 0.001 M CaCl2 and 2 ml of IgG-sepharose (Sigma) was added. After incubation for 5 two hours in the cold, the resin was spinned down. The supernatant was removed and diluted 5-fold with 0.02 M
Tris-HCl, 0.15 M NaCl, 0.001 M MgCl2, 0.001 M CaCl2.
Fifty microliters of this neutrophil lysate was then added to LM2-coated wells and incubated for 2 hours at 10 room temperature to capture the CDllb/CD18 integrin.
After washi~g with PBS, the plates were stored at -20 C.

In one type of assay, b;nAir~ of NIF (NIF-lFL and isoforms or engineered variants) to LM2/CDllb/CD18 coated plates was detected with the 3D2-HRP conjugate.
15 Samples con~s~in;r~q NIF were diluted in PBS contAining 0.1% (w/v) Skim-milk, 0.001 M MgCl2, 0.001 M CaCl2. After 2 hours incubation at room temperature, the wells were washed three times with 200 ~Ll PBS cont~ining 0.001 M
MgCl2, 0.001 M CaCl2, 0.02% Tween-20 and 0.02%
20 thimerosal. Then, 100 ~l of an appropriate dilution (typically 2000-fold; in PBS containing 0.1% (w/v) skim-milk, 0.001 M MgCl2, 0.001 M CaCl2) of the 3D2-HRP
conjugate was added. After 1 hour, the wells were washed three times with 200 ~l PBS containing 0.001 M
25 MgCl2, 0.001 M CaCl2, 0.02% thimerosal and 0.02% Tween 20. Substrate for HRP (100 ,ul; prepared by dissolving 2.3 mg ortho-phenylenediamine in 11.6 ml 50 mM citrate, 100 mM disodium phosphate buffer pH 5 and adding 2 ,ILl of 35% H202) was then added to the wells. The reaction was 30 stopped (typically after 20 minutes) by addition of 10 - lLl of a 4 M sulfuric acid solution; the absorbance was read at 605 nm with a Thermomax plate reader (Molecular Devices).
Alternatively, binding of NIF (NIF-lFL, other NIF
35 proteins, and NIF mutants) to the LM2/CDllb/CD18 complex W094/14973 PCT~S93/12626 ~,~5~9~ 70 was measured by competition with biotinylated recombinant NIF-lFL produced in Pichia (see Example 12).
Samples contAin;ng a constant amount of biotinylated recombinant NIF-lFL and varying amounts of unlabeled recombinant NIF-lFL were prèpared in PBS containing 0.001 M MgCl2, 0.001 M CaCl2 and 0.1~ (w/v) casein (Difco Laboratories). A 100 ~1 aliquot of each sample was added to individual LM2/CDllb/CD18 coated wells and incubated for 2 hours at room temperature. Unbound material was removed by washing three times with 200 ~1 PBS containing 0.001 M CaCl2, 0.001 M NgCl2, 0.1% Tween 20 and 0.02% thimerosal. Retained biotinylated recombinant NIF-lFL was detected by in~l~h~ting first with 100 ~l of ExtrAvidin-phosphatase (Sigma) conjugate diluted in PBS contAining 0.1% casein. After 1 hour incubation at room temperature, unbound conjugate was removed by w~h~ng three times with PBS contAining 0.001 M CaCl2, 0.001 M MgCl2, 0.02% Tween 20 and 0.02%
thimerosal. Substrate for alkaline phosphatase (100 ~l of a solution cont~ini~g 5 mg ortho-nitrophenylphosphate in 1.8 ml 0.01 M diethanolamine, 0.5 mM MgCl2, pH 9.5) was added. After 30 minutes the wells were read at 405 nm with a Thermomax plate reader to quantitate bound biotinylated recombinant NIF-lFL.

Example 2 Isolation of Native Neu~Gphil InhibitorY Factor From Hookworm Lysate (A) Pre~aration of Hookworm Lysate Frozen canine hookworms were obtained from Antibody Systems (Bedford, TX). Hookworms were stored at -70C
until used for homogenate.
Hookworms were homogenized on ice~in homogenization buffer [0.02M Tris-HCl pH 7.4, 0.05 M NaCl, 0.001 M
MgCl2, 0.001 M CaCl2, 1.0 x 105M dithiothreitol, 1.0 x 10-5M E-64 Protease Inhibitor (CAS 66701-25-5), 1.0 x W O 94tl4973 PCTrUS93/12626 -lO~M pepstatin A (isovaleryl-Val-Val-4-amino-3-hydroxy-6-methyl-heptanoyl-Ala-4-amino-3-hydroxy-6-methyl-heptanoic acid, CAS 26305-03-3), 1.0 x 10-5M chymostatin (CAS 9076-44-2), 2.0 x 10-5M APMSF (amidinophenyl-methylsulfonyl fluoride-HCl), 5% (v/v) glycerol] using a Tekmar Tissuemizer homogenizer. The protease inhibitors E64, pepstatin A, chymostatin, and APMSF were obtained from Calbiochem (La Jolla, CA). Approximately 3-6 ml of homogenization buffer was used to homogenize each gram of frozen worms (approximately 500 worms). Insoluble material was pelleted by two sequential centrifugation steps: 40,000 X gm~ at 4C for 20 minutes followed by 105,000 x gm~at 4C for 40 minutes. The supernatant solution was clarified by pASsAge through a 0.2 mm cellulose acetate filter (CoStar).

(B) Concanavalin A S~h~Arose Chromatoaraphv of Hookworm LYsate Hookworm lysate (79 ml) was adsorbed to 16 ml of Concanavalin A Sepharose (Pharmacia) pre-equilibrated with Con A buffer [0.02 M Tris-HCl, pH 7.4, 1 M NaCl, 0.001 M CaCl2, o.oo1 M MnS04, 1 x 10-5 M dithiotreitol] by recycling it through a 1.6 x 8 cm column at a flow rate of 3 ml/min (90 cm/hour) for 2 hours. The column was at room temperature (24C) while the reservoir of lysate was maintained on ice throughout the ~ G~ed~ e. The column was subsequently washed with 80 ml of Con A
buffer. The Con A buffer in the column was displaced with buffer containing 0.5 M methyl-alpha-mannopyranoside and flow stopped for 30 minutes. Flow was then restarted at a flow rat~e of 0.5 ml/min (15 ~ cm/hour). Material that had inhibitory activity in neutrophil function assays was eluted *ith approximately three column volumes of Con A buffer contAining 0.5 M
methyl-alpha-mannopyranoside (CAS 617-04-09). The yield WO ~/14973 PCT~S93/12626 2~S~S99 ~ -~ 72 of neutrophil adhesion inhibitory activity in this step was approximately 38%.
Figure 1 depicts Concanavalin A Sepharose chromatography of the hookworm lysate performed as described above. Absorbance at 280 nm was plotted as a function of time.

(C) Molecular Sieve Chromatoara~hY Using Superdex 200 Active fractions eluted from immobilized Concanavalin A (see step (B) above) and concentrated by ultrafiltratjon at 4C using an Amicon stirred cell equipped with a 10,000 dalton cut-off membrane (YM10), then 5-20 ml of the concentrate were loaded on a 2.6 cm x 60 cm column of Superdex 200 prep (Pharmacia) attached in series with an identical column (combined dimensions of 2.6 x 120 cm). Both columns were pre-equilibrated with 0.01 M potassium phosphate, pH 7.35, 0.150 M NaCl, l x 105 M dithiotreitol at 24C. The chromatography was conducted at a flow rate of 1.5 ml/min; anti-adhesion activity typically eluted 395-410 ml into the run (K.v of 0.46, see Fig. 2). This elution volume would be expected for a globular protein with a molecular mass of 50,000. The yield of neuL~Gphil function inhibitory activity in this step was typically 70-80~. If the ionic strength of the chromatography buffer employed was decreased to 0.01 M sodium phosphate, pH 7.00 and 10%
(v/v) glycerol added, the activity eluted substantially earlier (K~v = 0.34) suggesting that under such conditions the protein either aggregates or changes its conformation (assuming a larger Stoke's radius).
Figure 2 depicts Superdex 200 Chromatography of Concanavalin A-Purified Hookworm Lysate. Absorbance at 280 nm is plotted versus elution volume~. Active fractions eluted from immobilized Concanavalin A (see step (B) above) and concentrated by ultrafiltration at 40C using an Amicon stirred cell equipped with a 10,000 W094/14973 PCT~S93112626 dalton cut-off membrane (YM10), then 5-20 ml of the concentrate were loaded on a 2.6 cm x 60 cm column of Superdex 200 prep (Pharmacia) attached in series with an identical column (combined dimensions of 2.6 x 120 cm).
Both columns were pre-equilibrated with 0.01 M potassium phosphate, pH 7.35, 0.150 N NaCl, 1 x 10'5 M
dithiotreitol at 24C. The chromatography was conducted at a flow rate of 1.5 ml/min; activity eluted 395-410 ml into the run (~v of 0.46).

1o (D) Ceramic-HYdroxyapatite Chromatoara~hY
Material purified by molecular sieve chromatography was concentrated five-fold by ultrafiltration using an Amicon stirred cell equipped with a 10 kilodalton cut-off membrane at 4C and then diluted ten-fold with water. The desalted sample was loaded on a 0.8 x 10 cm column of ceramic hydLoxyapatite ("HA") (Pentax, American International Chemical, Inc., Natick, MA, 2 mm) equilibrated with 0.001 M potassium phosphate, pH 7.00, 1 X 10'5 M CaCl2, 1.0 x 10'5 M dithiothreitol at 24C. The loading was conducted at a flow rate of 0.8 ml/min (95.5 cm/hour). The column was developed with a 50 ml linear gradient of potassium phosphate ranging from 0.001 M to 0.0375 M at a flow rate of 0.5 ml/minute. Neutrophil inhibitory activity eluted sharply at 0.025 M potassium phosphate and then trailed to 0.0325 M potassium phosphate (fractions 37 to 48). The yield of activity in this step was approximately 48%.
Figure 3 depicts Ceramic IIydLoxylapatite Chromatography of Superdex/Concanavalin A-Purified Hookworm lysate plotting absorbance at 280 nm and ~ potassium phosphate concentration versus fraction number. Neutrophil inhibitory activi~y eluted in fractions 37 to 48.

(E) Reverse Phase HPLC

W094/14973 PCT~S93/12626 -Hookworm lysate fractionated by chromatography on Concanavalin A Sepharose, Superdex, and ceramic hydroxylapatite (-100 mg) was loaded on to a 0.48 x 15 cm column of 300 a..y~L~vm C4 (Vydac) which was then developed with a linear gradient of 0-60% acétonitrile in 0.1% trifluoroacetic acid at 1 ml/minute with a rate of 1% change in acetonitrile/minute. NeuLlo~hil inhibitory activity typically elutes between 41 and 45%
acetonitrile, the activity corresponding with a broad peak.
Figure~4 depicts the results of reverse phase HPLC
of the Neutrophil Inhibitory Factor. Inhibitory activity eluted between 43 and 45% acetonitrile, the activity corresponding with a broad peak at 43-45 minutes.

Table I
Summary of Examle Pur; f; cation FR~CTIONATION PROTeIN PLRCLNT SPECIFIC FOLD
STLP (mg)ACrlVI~ ACrlVlTY PURIP.
2 0 EXl`RA~ S28~ 100 0.2 ConA ~LUATE 21.738 1.8 9 SUPERDEX POOL 1.5 25 16.7 88 HYDROXYAPATITE 0.3 12 40.0 200 POOL

2 5 Examle 3 Isolation of the Neutrophil Inhibitory Factor From Hookworm Lysate Usina Prearative Isoelectric Focusinq Hookworm lysate was partially fractionated and desalted by molecular sieve chromatography on a 2.6 cm x 60 cm column of Superdex 200 prep (Pharmacia) attached in series with an identical column (combined dimensions of 2.6 x 120 cm). Both columns were pre-equilibrated with 0.01 M sodium phosphate, pH 7.00, 10% (v/v) glycerol at 24C. Adhesion inhibiting fractions eluting WO ~/14973 PCT~S93112626 2~5~5~

at 350-370 ml were diluted to 55 ml by the addition of 1.4 ml of 40% Biolyte 3-10 ampholyte (BioRad) and 10%
(v/v) glycerol. This mixture was focused with a ; constant power of 12 W for 5 hours at 4C in a Rotofor preparative isoelectric focusing prep cell (BioRad).
Twenty fractions were harvested; inhibitory activity was detected in fractions 6-9, corresponding to an isoelectric point of 4.5. The overall yield of inhibitory activity for this step was approximately 30%.

Example 4 ~
Ion Exchange Chromatography Hookworm lysate fractionated by molecular sieve chromatography on Superdex 75 (Pharmacia) was mixed with an equal volume of Mono Q buffer tO.02 M Tris-HCl, pH
7.5] and loaded on to a 0.5 x 5.0 cm Mono Q anion exchange column (Pharmacia) equilibrated with Mono Q
buffer at a flow rate of 1 ml/minute (306 cm/hour). The column was then developed with a l; ~eAr gradient of 0-0.5 M NaCl in column buffer at 0.5 ml/minute (153 cm/hour). NeuLL~hil inhibitory activity consistently eluted at 0.4 M NaCl. The overall yield of inhibitory activity for this isolation was about 2-5%.

Example 5 SDS-PolYacrYlamide Gel Ele~L~u~horesis The protein composition of hookworm lysate and fractionated lysate was analyzed by sodium dodecyl sulfate polyacrylamide gel eleu ~L ophoresis (SDS-PAGE) (Laemmli, U.K. 1970, Nature 227, 680) after silver staining (Morrisey, J.H. 1981, Anal. Biochem. 117, 307).
~ 30 Samples were mixed with an equal volume of 20% glycerol, 5% SDS, and 0.125 M Tris-HCl, pH 6.8 a~d placed in a boiling water bath for 5 minutes. Samples were subsequently applied onto 10% SDS polyacrylamide slab . W094/14973 PCT~S93tl2626 21$~599 76 gels of 0.75 mm thickness and subjected to electrophoresis for 2 hours at constant voltage (125 V).
Figure 5 depicts the results of SDS polyacrylamide gel electrophoresis. Samples were applied to a 10~
polyacrylamide slab gel (Novex, La Jolla, CA). Lanes 1-10, left to right, are (1) molecular weight st~n~Ards;
(2) molecular weight st~Ards; (3) HPLC pool of HA
fractions #37-41, non-r ~llc ~; (4) blank; (5) HPLC pool of HA fractions #37-41, reduced; (6) blank, (7) HPLC
pool of HA fractions #37-41, reduced, (8) HPLC pool of HA fractions~#37-41, non-reduced; (9) HPLC pool of HA
trailing fractions #42-48, non-r~A~lc~, (lO)molecular weight st~ ~ds. The molecular weight st~nA~rds used were: myosin, 200,000 (rabbit muscle);
beta-galactosidase, 116,300 (E. oli); phosphorylase b, 97,400 (rabbit muscle); bovine serum albumin, 66,300;
glutamic dehydrogenase, 55,400, (bovine liver); carbonic anhydrase, 31,000, (bovine erythrocyte); trypsin inhibitor, 21,500, (soybean).
Following the last step of the isolation procedure (reverse phase HPLC) only a single diffuse band with an apparent molecular weight ranging from 33,000 to 47,000 was observed upon SDS-PAGE (see Fig. 5). When 50 mM
dithiothreitol was added to the sample prior to boiling, the diffuse band migrated with an estimated molecular weight of 43,000 to 54,000.

Example 6 Laser-Desorption Time-of-Fliqht Mass S~ectrometrY of the Isolated Neutro~hil InhibitorY Factor The estimated mass for the NIF isolated as described in Example 2(E) was determined using laser-desorption time-of-flight mass sp~ctrometry.
A 1 ml aliquot of the sample was diluted with an equal volume of a saturated solution of 3,5-dimethozy-4-hydroxy-cinnamic acid dissolved in 30%

W094/14973 PCT~S93/12626 2~ ~259g aqueous CH3CN, 0.1% TFA. The diluted sample was spotted onto a copper sample stage and allowed to air dry. Mass analysis was performed using a Shimadzu LAMS-SOKS laser desorption time of flight mass spectrometer (Shimadzu Corp., Kyoto, Japan). Ionization of the sample was accomplished by focusing 500 laser pulses (355 nm, pulse width <5 nsec) from a Nd-YAG laser (Spectra-Physics, Inc., Mt. View, CA) onto the sample stage. The resulting ions were accelerated into the mass spectrometer by a 5 kV potential. Calibration of the instrument ~as accomplished using stAn~rd proteins of known mass.
Figure 6 depicts the results of laser-desorption time-of-flight mass spectrometry of the isolated neutrophil adhesion inhibitor. Five picomoles of the purified neutrophil function inhibitor was analyzed with a laser desorption time-of-flight mass spectrometer. The estimated mass was determined as 41,200. A small fraction of the sample had a mass of 82,400; this was interpreted to be a dimer.

Example 7 Neutro~hil Inhibitory Factor is a Glyco~-G~ein Purified NIF (prepared according to Example 2(E)) (-2 mg) was electrophoresed in a 10% SDS polyacrylamide gel and the resolved protein transferred by Western blotting (Towbin, et al., 1979 Proc. Natl. Acad. Sci.
(USA) 76, 4350-4354) to a Zeta-Probe0 nitrocellulose membrane (BioRad, Emeryville, CA). The membrane was treated as described in the instructions to the GlycoTrack~ Kit (Oxford GlycoSystems, Rosedale, NY) to ~ oxidize carbohydrates to aldehydes which were then reacted with biotin-hydrazide leading t0 incorporation of biotin into any carbohydrate present. Biotinylated carbohydrate was subsequently detected by reaction with a streptavidin-alkaline phosphatase conjugate.

W094tl4973 PCT~S93/12626 2~25 99 ~ : 78 Visualization was achieved using a substrate which reacts with alkaline phosphatase bound to glycoproteins on the membrane, forming a colored precipitate.
Neutrophil Inhibitory Factor was StA i n~ using this method, demonstrating that it contained carbohydrate and is therefore a gly~o~oLein.

Examle 8 Organic Extraction of the Hookworm Lysate One milliliter of hookworm homogenate known to have inhibitory activity in the neu~Lo~hil-plastic adhesion assay was extracted by vort~xing 1 minute with 1 ml of a chloroform/methanol (2:1) mixture in a 15 ml glass Corex test tube. The organic layer was removed and dried under a stream of nitrogen gas. Residual lipids were resusp~n~ in 0.5 ml HSA assay buffer by sonication for 2 minutes (Branson Model 1200, nAnhllry~ CT). Resuspended lipids had no inhibitory activity in the neutrophil-plastic adhesion assay when tested at a final dilution of 1:2.

Example 9 Production And Determination Of The Amino Acid Seauence Of Peptide Fraaments Of Ne~LLo~hil Inhibitory Factor Samples of NIF were obtAin~ as described in Example 2. Two separate volumes, each contAining approximately 10 mg NIF, were first degassed on a Speed Vac until the samples were frozen and then lyophilized.
The dried samples were resuspen~ in 50 mM
N-ethylmorpholine, pH 8.5, and digested with either endoproteinase AspN (Boehringer Mannheim, Indianapolis, IN), Lys C (Boehringer ~Annh~im~ Indianapolis, IN) or trypsin (Worthington, Freehold, NJ) at ~ substrate to enzyme ratio of 25:1. Tnc~hAtion was at ambient temperature for 24 hours and a small amount of isopropanol was added to the digestion mix to prevent W094/14973 PCT~S93/12626 21~99 microbial contamination. At the end of the digestion, the samples were d~gAs~^~ on a Speed Vac and dried by lyophilizing. The digestion mixtures were resuspended in 6M guanidine/HCl for fractionation of peptides by reversed phase HPLC (RP HPLC). Peptides were isolated by RP HPLC on a ToyoSoda 120T C18 (4.5 X 250 mm) column using an LKB HPLC system with Kratos (ABI, Foster City, CA) detectors. The column was developed with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid (TFA). The gradient was from 5 to 54% acetonitrile over 120 minutes-~at a flow rate of 0.5 ml/minute. Peptide peaks monitored by A~ and ~ were collected using an LKB SuperRac with calibrated peak detection. The collected fractions were neutralized with ammonium carbonate, 20 mg SDS was added, and the fractions dried under N2 before sequencing. Peptides were se~nce~ on a 470A/120A/9OOA gas phase sequencer (ABI, Foster City, CA). Residue identification was performed manually by analysis of the HPLC chromatograms and quantification of the PTH residues was performed by online analysis on the 900A computer. Cysteine residues were not detected in this analysis because the protein had not been alkylated. In experiments in which the protein was digested with trypsin, the protein was alkylated with vinylpyridine before fragmentation, thereby permitting the detection of cysteine in the tryptic fragments.
Aspartic acid and tryptophan residues were identified but not quantitated because background peaks overlapped the PTH residues in the HPLC elution. The initial ~ 30 yields ranged from 1 pmole to 10 pmole and the repetitive yield was usually between 92 and 95%. Figure ~ 7 depicts the amino acid sequences that were obtained from the proteolytic fragments. In Fig~re 7, positions enclosed in parentheses were not determined with absolute certainty. Abbreviations for amino acids beginning with a capital letter were observed in higher W094/14g73 PCT~S93/12626 :, 2152~99 80 yield and are preferred in these cases. The abbreviation Xxx indicates an undetermined amino acid at that position, since no specific amino acid was identified during Edman degradation of the peptide. See Scarborough et al. J. Biol.`Chem 266:9359, 1991.; Perin et al., J. Biol. Chem. 266:3877, 1991.

Example 10 Cloninq and Sequencin~ of NeU~L~hi1 InhibitorY Factor from Hookworm NIF was ~loned from a canine hookworm cDNA library, constructed as follows: Total RNA was isolated from whole hookworms by guanidium thiocyanate extraction (McDonald et al., Meth. Enzymol. 152:219 (1987)).
Poly(A)+ RNA was purified from 500 mg of total hookworm RNA using oligo d(T) cellulose affinity chromatography (PolyA Quik; Stratagene, La Jolla, CA). Double stranded cDNA was synthesized from poly(A)+ RNA using random hexamer primers and avian myoblastosis virus (AMV) reverse transcriptase (Amersham, Arlington Hills, IL).
cDNA fragments larger than 1 kilobase pairs were purified on a 6% polyacrylamide gel and ligated to EcoRI
linkers (Stratagene) using st~ rd procedures.
Linkered cDNA was ligated into lambda gtlO (Stratagene, La Jolla, CA) and packaged using Gigapack Gold II
(Stratagene).
Double stranded cDNA probes for hookworm NIF were generated by polymerase chain reaction from hookworm RNA
using primers derived from NIF peptide sequences. The sequences obtained for two NIF peptides (see Fig. 7), T-20 (Leu-Ala-Ile-Leu-Gly-Trp-Ala-Arg) and T-22-10 (Leu-Phe-Asp-Arg-Phe-Pro-Glu-Lys), were used to design primers 30.2 and 43.3.RC, respectively. ~The sequences of 30.2 and 43.3.RC were 5'-CTCGAATTCT(GATC)GC(ATC)AT(ATC)(CT)T(GATC)-GG(ATC)TGGG
C-3' and WO ~/14973 PCT~S93112626 S'-CTCGAATTCTT(TC)TCTGG(GA)AA-(GA)CG(GA)TC(GA)AA-3', respectively. Bracketed positions represent redundant nucleotides. Single stranded cDNA was synthesized by priming 1 mg of hookworm poly(A)+ RNA (preparation described above) with random heY~mlsleotides and extending with AMV reverse transcriptase (Amersham, Arlington Hills, IL). One twentieth of the reaction product was amplified using the PCR GeneAmp kit (Perkin Elmer, Norwalk, CT), with 400 pmol of each of 30.1 and 43.RC (manufactured by Res^~rch Genetics, Huntsville, AL), on a Perkin Elmer DNA Thermal Cycler. PCR
conditions were: cycles 1-2, denaturation at 94C for 2 minutes, annealing at 58C for 2 minutes and elongation at 72C for 2 minutes; cycles 3-42, denaturation at 94C
for 45 seconds, An~eAling at 58C for 45 seconds and elongation at 72C for 2 minutes. The -430'base pair amplification product, referred to as the 30.2/43.3.RC
fragment, was separated from reaction contaminants by electroelution from a 6% polyacrylamide gel (Novex, San Diego, CA). The 30.2/43.3.RC fragment was labelled with [~-32P]-dCTP (Amersham) using random primer labelling (Stratagene, La Jolla, CA); labelled DNA was separated from unincorporated nucleotides using a ChromaSpin-10 column (Clontech, Palo Alto, CA).
Prehybridization and hybridization conditions were 6X SSC (SSC: 150 mM NaCl, 15 mM trisodium citrate), 0.02 M sodium phosphate pH 6.5, 5X Denhardt's solution, 0.5% (w/v) SDS, 0.01 M EDTA, 100 mg/ml sheared, denatured salmon sperm DNA, 0.23% dextran sulfate, 50%
- 30 formamide. Prehybridization and hybridization were at 42C, and the filters were washed for 20 minutes with 0.2X SSC at 60C after two prewashes with 2X SSC for 15 minutes. The filters were exposed over~ight to X-ray film with two intensifying screens at -70C.
Approximately 300,000 recombinant phage of the random primed hookworm library (unamplified) were WO94/14973 PCT~S93112626 2 ~S2599 ~ 82 screened with the 30.2/43.3.RC NIF PCR fragment. About 120 recombinant phage hybridized to this probe, of which seven were isolated for nucleotide sequencing analysis.
Double stranded sequencing was effected by subcloning the EcoRI cDNA fragments contained in these phage isolates into pBluescript II vector (Stratagene, La Jolla, CA). DNA was sequenced using the Sequenase version 2.0 kit (U.S. Biochemical, Cleveland, OH) and synthetic oligonucleotide primers.
The NIF phage isolates contained DNA that encoded polypeptide~ that bore striking resemblance to the amino acid sequences obtained for purified NIF (see Figure 7).
Figure 8 depicts the nucleotide sequence of the coding region of NeuLLGphil Inhibitory Factor cDNA (clone lFL) and its predicted amino acid sequence. A single isolate, NIF-lFL, encoAP~ an open r~ g frame of 825 nt, initiating with a methionine and terminating with a TGA stop codon (Figure 8). The NIF polypeptide encoded by NIF-lFL is 274 amino acid residues with a calculated molecular weight of 30,680 daltons. Figure 9 depicts the alignment of the predicted amino acid sequences of several Neutrophil Inhibitory Factor isoform clones.
Each line of sequence represents the corresponding sequence segments of the various clones isolated. Each segment is identified by its clone designation (e.g., lFL, 3P, 2FL, 3FL, 4FL, 6FL and lP). The complete amino acid sequence of clone lFL is listed in standard three-letter amino acid code at the top of each sequence segment. Clones having the same amino acid in a given position as clone lFL are denoted by ".". Amino acid substitutions are indicated by the appropriate three-letter code. "---" indicates a space inserted to maintain alignment of the sequences. T~e carboxy termini of the lFL and lP sequences are denoted by an asterisk. The other six NIF phage isolates encoded partial NIF polypeptides; that is they did not contain WO ~/14973 PCT~S93112626 either an N-terminal methionine residue or a C-terminal stop codon, as compared to the NIF-lFL polypeptide (Figure 9). These partial NIF isolates comprised six predicted NIF isoforms that were significantly similar to, but not identical to the prototypical NIF-lFL
polypeptide.

Example ll Ex~ression of Functional Recombinant Neutrophil Inhibitory Factor by Mammalian Cells (A) Transi~nt Exoression in COS-7 Cells.
The segment of DNA enco~in~ the NIF-lFL isoform was amplified from the original ~gtlO isolate DNA using unique primers for the 5'- and 3'-ends of the roAing region.
The 5'-primer was composed of a restriction endonuclease site (EcoRl), a ro~cencus ribosome binding site (Kozak, N., Cell 44: 283 (1986)), the ATG
initiation codon of NIF and the s~lcceeAing 6 codons of the gene. The 3'-primer was composed of a unique nucleotide sequence to the 3'-side of the TGA
termination codon of NIF and a restriction endonuclease site (EcoRl). The nucleotide sequences of the 5'- and 3'-primers were 5'-ACC-GAA-TTC-ACC-ATG-GAG-GCC-TAT-CTT-GTG-GTC and 5'-CTG-GAA-TTC-TCG-CTT-ACG-TTG-CCT-TGG-C, respectively.
Five microliters of the lambda plaque suspended in l ml dilution buffer were used as template DNA.
Amplification was accomplished using the PCR GeneAmp kit (Perkin Elmer, Norwalk, CT), with 400 pmol of each of the 5'- and 3'-primers (manufactured by Research Genetics), on a Perkin Elmer DNA Thermal Cycler. The PCR conditions were: cycle l, denatura~ion at 97C for l minute, primer annealing for l minute at 37C, ramp from 37C to 72C in 2 minutes, and amplification for 2 minutes at 72C; cycles 3 and 4, denaturation at 94C

W094/14973 PCT~S93/12626 2~ 99 84 for 1 minute, primer annealing for 1 minute at 37C, ramp from 37C to 72C in 2 minutes, and amplification for 2 minutes at 72C; cycles 5 through 34, denaturation at 94C for 1 minute, primer annealing for 1 minute at 45C, and amplification for 2 minutes at 72C. -~
The amplification product (887 bp) was separatedfrom reaction contaminants using a ChromaSpin 400 column (Clontech Laboratories, Inc. Palo Alto , CA). The ends of the amplification product were trimmed with the restriction~endonuclease EcoR1 and the resulting fragment of DNA (875 bp) ligated into EcoR1-digested plasmid pSG5 (Stratagene, La Jolla, CA) using st~Ard tec~n;ques. The resulting ligation mixture was used to transform SUREH competent cells (Stratagene, La Jolla, CA).
An isolate containing the 875 bp insert in the proper orientation (5'-end of the co~in~ region proximal to the pSG5 SV40 promoter) was grown in 250 ml Circle Grow~ (Biolo, San Diego, CA) with 50 mg/ml ampicillin and plasmid DNA was prepared using a Magic Maxi Prep~
DNA purification system (Promega, Madison, WI). Ten micrograms of purified plasmid DNA was transferred into 3.5 x 106 COS7 cells (ATCC No. CRL 1651) by electroporation (0.4 cm ele~L~upGLation cell, 325 V, 250 F, infinite resistance, 0.5 ml cells at 7 x 106/ml in Hepes buffered saline, pH 7.0, 4C). After electroporation the cells were allowed to stand on ice for 2 to 3 minutes before dilution with 14 ml warm DMEM:RPMI 1640 (1 to 1 ratio) supplemented with 10%
fetal bovine serum prewarmed to 37C. The cells were placed in 100 mm cell culture ~ish~s and incubated at 37C with 8% CO2. Cell culture superna~nt fluid was removed at 1, 2 and 3 days after plating and assayed for NIF activity.

WO ~/14973 PCT~S93/12626 -(B) Detection and Ouantitation of Neutro~hil Inhibitory Factor Activity in Cell Culture Medium.
15 ml of cell culture fluid was harvested from ele~Lo~orated COS7 cells (pSG5/NIFlFLCRl). When assayed directly using the neutrophil-plastic adhesion assay (Example l(C)), this fluid exhibited neutrophil inhibitory activity to dilutions as great as l:8. An IC50 at approximately l:14 was determined using the hydrogen peroxide release assay (Example l(E)). No activity was observed using cell culture fluid harvested from COS7 cells ele~Lu~o~ated with a control expression plasmid (pCAT; Promega, Madison, WI).

(C) Stable Ex~ression in CHO Cells (l) Preparation of Plasmid DNAs The NIF-lFL insert in the pSG5 construct described above in section (A) was excised by digestion with the restriction endonuclease EcoRI. The 875 bp NIF-lFL
fragment was gel purified (Magic PC Prep, Promega, Madison, WI) and ligated into EcoRI digested pBluescript II KS (Stratagene, La Jolla, CA) using stAn~Ard techniques. The resulting ligation mixture was used to transform SURE~ competent cells as described by the supplier (Stratagene, La Jolla, CA). Transformed cells were plated on LB agar containing IPTG and X-gal (Sambrook, Fritsch, and Maniatis, MoleclllAr Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, pp. l.85 to l.86). White colonies were screened for plasmids contA;ninq the NIF-lFL insert in the proper orientation by digesting plasmid DNA with BamHI; those colonies harboring a plasmid that yielded a 200 bp ~HI
fragment were retained. Plasmid was prepared from one of these colonies (Magic Maxi Prep, ProMega, Madison, WI) and digested with HindIII and NotI to yield a NIF-lFL fragment with a HindIII overlap on the 5'-end and a NotI overlap on the 3'-end. This DNA fragment was WO94/14973 PCT~S93/12626 ?~P593 gel purified and ligated into HindIII-NotI digested pRC/CMV (Invitrogen, San Diego, CA). The resulting ligation mixture was used to transform SURE~ competent cells. Milligram quantities of pRC/CMV-NIF-lFL were prepared using the Magic Maxi Prep kit.
The plasmid pLTRdHFR26 (Mol. Cell. Biol. 3:32-43 (1983), Nature 275:617-623 (1978)) in the E. coli strain RRI was purchased from the ATCC (American Type Culture Collection, Rockville, MD). Plasmid DNA was purified from a chloramphenicol amplified culture (Sambrook, Fritsch, an~ Maniatis, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, p.
1.33) using the Magic Maxi Prep kit.
.

(2) Transfection of CHO Cells Ch;nPs~ hamster ovary (CHO) cells harboring a defect in the dihydrofolate reductase gene (dhfr) were obtained from the ATCC (catalog number: CRL 9096) and grown in a 1:1 mixture of RPMI 1640 and DMEM media (Irvine Scientific, Santa Ana, CA) supplemented with 10%
FBS, 10 mM HEPES, essential amino acids, nonessential amino acids, 5 x 105 M ~-mercaptoethanol, 10 mM sodium pyruvate, and 2 mM glutamine (combo medium). The CHO
cells were transfected using the Calcium Phosphate Transfection System following the manufacturer's instructions (Gibco BRL, Gaithersburg, MD) in 10 cm cell culture dishes at the following ratios: 1 x 106 cells, 20 g pRC/CMV-NIFlFL DNA and 5 g pLTRdHFR26 DNA. The cells were incubated for 12 hr at 37'C in the presence of the co-precipitated DNAs.

(3) Selection and Am~lification of CHO-NIFlFL
Clones The adhered cells were washed once with fresh combo medium and placed under combo medium containing 500 g/ml of the antibiotic G418 (Gibco BRL, Gaithersburg, MD) for W094/14973 PCT~S93/12626 ~_ 21~2599 two weeks at 37-C to select for those clones exhibiting neomycin resistance. The G418 resistant cells were trypsinized from the culture ~;ches using st~AArd techn;ques and washed once with fresh combo medium containing 500 g/ml G418. The washed cells were allowed to attach to 10 cm cell culture ~;ch~s (1 x 106 per dish) and covered with combo medium contA;n;ng 500 g/ml G418 and 10, 20, 40, 60, or 80 nM methoLLexate (Sigma, St.
Louis, MO). After two weeks incubation at 37-C, the dishes were examined for colonies and the culture supernatant f luids assayed for NIF activity using the calcein assay for neuL,o~hil adhesion. The cells in dishes exhibiting NIF activity were trypsinized and washed as before and plated to obtain single colonies.
One single cell isolate, designated 8F5, expressing NIF
activity was chosen for further methotrexate amplification.
8F5 cells were grown to confluence in a 10 cm cell culture dish, detached with trypsin and washed as before. The cells were diluted with combo medium containing 500 g/ml G418 and 1 x 106 cells were placed in 10 cm culture dishes. The adhered cells were covered with combo medium containing 500 g/ml G418 and 40, 80, 160, 320, or 640 nM methotrexate. Again, after two weeks incubation at 37-C, the ~;ches were examined for colonies and supernatant fluids assayed for NIF
activity. Cells were released from the plates by trypsin treatment as before. The pool of cells obtained from the 320 nM methotrexate dish was selected for further use and single cell isolates were obtained.
Three successive rounds of single cell isolation were performed on each colony to ensure clonal purity. The final cell line, designated 8F5-lE6-8Cl~lG8, produces NIF at greater than 50 g/ml in the presence of 500 ~g/ml G418 and 320 nM methotrexate.

W094/14973 PCT~S93/12626 25 9g ~ ~ 88 (D) Fractionation of Neutrophil InhibitorY Factor Activity bY Chromatography on Immobilized -Concanavalin A.
Five ml of COS7(pSG5/NIFlFLCRl) cell culture fluid was mixed with an equal volume 0.02 M bis Tris-propane-HCl, pH 7.3, 1 M NaCl, 0.001 M CaCl2, 0.001 M MnS04 and loaded onto a one ml column of ConcAn~valin A
Sepharose (Pharmacia, Piscataway, NJ) equilibrated with the same buffer. The sample was cycled through the column in a closed loop for 1 hour at 2 ml/minute at 20C. The ~olumn was subsequently washed with 5 ml of 0.02 M bis Tris-propane-HCl, pH 7.3, 1 M NaCl, 0.001 M
CaCl2, 0.001 M MnS04. The buffer resident in the column - - was displaced with buffer contA~n;ng 0.5 M
methyl-alpha-mannopyranoside and flow stopped for 15 minutes. Flow was restarted at 1 ml/minute and approximately 11 ml of sugar-cont~in;ng eluate collected. The eluate was dialyzed 18 hours against 1 liter 10 mM potassium phosphate, pH 7.35, 150 mM NaCl at 4C and concentrated to 1.1 ml using an Amicon centrifugal conce"LLator equipped with a 10,000 molecular weight cut-off membrane (CentriPrep 10, Amicon, Beverly, MA). When assayed by the neutrophil-plastic adhesion assay (Example l(C)), this sample exhibited substantial activity at a dilution of 1:16, indicating that a significant portion of the neutrophil function inhibitor activity present in the cell culture fluid binds to immobilized Concanavalin A.
This behavior is identical to that observed for crude extracts of Anc~lostoma caninum (Example 2(B)) and is consistent with the inhibition resulting from the synthesis and secretion from transfected mammalian COS7 cells of a glycoprotein that acts as an~inhibitor of neutrophil function.
As a control, 5 ml of COS7 cell culture medium from cells electroporated in the absence of DNA was W094/14973 PCT~S93/12626 '- 2152599 chromatographed on Concanavalin A Sepharose in the same manner as described above. No activity was observed after Concanavalin A-Sepharose chromatography using the neutrophil-plastic adhesion assay (Example l(C)).

(E) Fractionation of Neutrophil InhibitorY Factor Activity bY Anion Exchange Chromatoqraphy using POROS II O/M.
Five ml of COS7(pSG5/NIFlFLCR1) cell culture fluid was dialyzed 18 hours against one liter of 10 mM bis Tris-propane~HCl, pH 7.0 at 4C and loaded at 3 ml/minute onto a 0.46 x 10 cm column of Poros II Q/M
(PerSeptive Biosystems, Inc., League City, TX) equilibrated with the same buffer. The column was washed with one column volume of equilibration buffer and developed with a li~eAr gradient of sodium chloride from 0 to 0.5 M over 14.4 column volumes collecting 2 ml fractions. Significant activity in the neutrophil-plastic adhesion assay (Example l(C) was detected in fractions 17 and 18, corresponding to about 0.45 M NaCl. When fractions were concentrated twenty-fold using centrifugal concentrators equipped with a 10,000 MWCO membrane (Amicon MicroCon 10, Beverly, MA), substantial activity was found in fractions 16-19.
Neutrophil inhibitory factor present in extracts from AncYlostoma caninum elutes likewise from an anion exchange column (Mono Q, Pharmacia, Piscataway NJ) at 0.4 M NaCl (Example 4).

Example 12 Ex~ression of Functional Recombinant NeuLLu~hil Inhibitory Factor in Pichia ~astoris (A) Description of the Pichia shuttle/expression vector.

W094/14973 PCT~S93tl2626 The Pichia strain GTS115 (his4)(Stroman, D.W. et al., U.S. Patent No. 4,855,231 (August 8, 1989)) and the E. coli-Pichia shuttle vectors pHILSl and pHILD5 referred to hereafter are part of the Pichia yeast expression system licensed from the Phillips Petroleum Company (Bartlesville, OklAhoma).
All of the Pichia manipulations were performed essentially as described for Saccharomyces cerevesiae in Gene Expression Technology, pp.231-471, Academic Press, New York, (D.V. Goeddel, edit. 1991) and in Stroman, D.W. et al.,~ US Patent No. 4,855,231 (August 8, 1989).
The pHIL7SP8 vector used to direct expression of NIF in P. ~astoris was assembled from pHILSl and pHILD5 and from synthetically generated fragments. The pHIL7SP8 plasmid contA i ne~ the following elements cloned onto pBR322 seql~ncec:
1) 5' AOXl, about 1000 bp segment of the P.
pastoris alcohol oxidase 5' untranslated and promoter sequences (see Stroman, D.W. et al., U.S. Patent No.
4,855,231 (August 8, 1989) the disclosure of which is incorporated herein by reference).
2) The PHOl P. pastoris secretion signal.
3) A l9-amino acid synthetic pro-sequence fused to the PHOl signal. This ~LO sequence represents one of the two l9-aa pro-sequences designed by Clements et al.,(1991. Gene, 106:267-272) on the basis of the yeast alpha-factor leader sequence.
4) A synthetic multi-cloning site.
5) 3' AOXl, about 256 bp segment of the aoxl terminating sequence (see Stroman, D.W. et al., U.S.
Patent No. 4,855,231 (August 8, 1989) the disclosure of which is incorporated herein by reference).
6) P. ~astoris histidinol dehydL~enase gene, his4, contained on a 2.4 kb fragment to complement the defective his4 gene in the host GTS115 (see Stroman, D.W. et al., U.S. Patent No. 4,855,231 (August 8, 1989) WOg4/14973 ~ PCT~S93/12626 the disclosure of which is incorporated herein by reference).
7) Region of 3' AOX1 untranslated DNA sequence, which together with the 5' AOX1 region is ne~eCcAry for site-directed integration (see Stroman, D.W. et al., U.S. Patent No. 4,855,231 (August 8, 1989) the disclosure of which is incorporated herein by reference).

(B) Construction of pHIL7SP-NIcl/DHIL7SP-NIc10 and exoression in Pichia.
The segment of DNA encoA i ng NIF was PCR-amplified from a sub-clone of NIF-lFL in BluescriptII (Stratagene, La Jolla, CA) using unique primers for the 5'- and 3'-ends of the coding region.
The 5'-primer contA;n~ no restriction endonuclease sites and corresponded to the region beginning at the 5'-end of proteolytically procesce~ NIF and the succ~ing 7 codons. The codon for the first residue of the mature NIF was altered from AAT to AAC (both codons~
translate to asparagine). The 3'-primer was composed of

8 codons at the 3' end of the coA; ng region, a TAA stop replacing the TGA stop of the natural gene, and three unique restriction endonuclease sites (HindIII, S~eI, and BglII). The se~len~e~ of the 5'- and 3'-primers used were 5'-AAC-GAA-CAC-AAC-CTG-AGG-TGC-CCG and 5'-CCT-CCT-CCT-AGA-TCT-AAG-CTT-ACT-AGT-TTA-TAA-CTC-TCG-G
AA-TCG-ATA-AAA-CTC, respectively.
Amplification was accomplished using 100 pmol of each primer, 2 units of Vent polymerase in lX Vent buffer (New England Biolabs, Beverly, MA), and 0.2 mM of each of dATP, dCTP, dGTP, and dTTP. One hundred nanograms of BluescriptII-contAi ni ng NIE-lFL were used as template DNA. The PCR conditions were the same for all ten cycles: denaturation at 95C for 1 minute, primer annealing at 60C for 1 minute, and amplification WO94/14973 PCT~S93/12626 for 1.5 minutes at 72C. The amplification product was purified as described above and digested with BqlII.
The amplification product was then ligated into StuI-BalII cleaved pHIL7SP8 using stAnAArd methods. The S ligation mixture was used to transform E.coli WK6, and ampicillin resistant clones were obtained on ampicillin plates. Based on restriction and DNA sequence analysis, correct insert sequences in two of the resulting plasmid clones, pHIL7SP-NIlcl and pHIL7SP-NIlc10, were selected to transform the P.pastoris yeast strain GTS115 (his4).
These vectors were digested with either Noti (targeting integration to the expression cassette in the AOX1 region) or Sall (targeting integration to the HIS4 locus).
The 4 restricted DNA preparations were introduced individually into Pichia by ele~L.GpGlation, essentially as described by Becker, D. and Guarente, L., Methods in Enzymology, vol. 194, pp. 182-189 (1991). Briefly, the cells were grown in YEPD medium at 30C to an OD~ of 1.3 to 1.5. The cells were pelleted at 4C (1500 x g for 5 minutes) and resuspended in 500 ml ice cold sterile distilled water. The cells were pelleted as above and resuspended in 250 ml ice cold distilled water. After the cells were pelleted again, they were resuspended in 20 ml ice cold 1 M sorbitol. After a final pelleting the cells were resusp~nAeA in 1 ml ice cold 1 M
sorbitol. Forty ~l cells in 1 M sorbitol were mixed with 5 ~1 of linearized DNA and the mixture transferred to an ice cold 0.2 cm gap ele~LrG~G,ation cuvette.
After 5 minutes on ice, the cells were pulsed at 50 uF, 1.5 kV/cm, and 200 n resistance. One ml of ice cold 1 M
sorbitol was added to the ~uveLLes and 100 to 500 ul of the cell suspension were spread on mini~al dextrose plates. The plates were incubated at 30C until colonies appeared. The transformation mix was plated on minimal dextrose (MD) medium to select for His+

W094l14973 PCT~S93/12626 Q ~
93 ~1~2~99 transformants. Subsequent selection for NIF expression was performed in shake flask cultures in minimal medium containing methanol as described in Stroman, D.W. et al., U.S. Patent No. 4,855,231 (August 8, 1989) (C) Detection and Ouantitation of Neutro~hil InhibitorY
Activity in Cell Medium.
Pichia cell supernatant (pHIL7SP-Nlc10) was obtained by centrifugation for 15 minutes at 1,800 x g~x from cells 48 hours following methanol induction and filtered through a 0.22 ~m cellulose acetate membrane.
The filtered cell supernatant solution was concentrated about 3-fold using centrifugal concentrators equipped - - with a 10,000 MWCO membrane (Amicon MicroCon 10, Beverly, MA) and desalted by gel filtration using a 1 x 10 cm column of G-25 Sephadex Superfine (Pharmacia, Piscataway, NJ). Using the neuL~G~hil-plastic adhesion assay (Example l(C)), the desalted supernatant solution (diluted 2x by gel filtration) exhibited neu~LG~hil inhibitory activity to dilutions as great as 1:640. No activity was observed using cell supernatant solution similarly harvested and treated from Pichia cells expressing a recombinant anti-thrombotic protein devoid of neutrophil inhibitory activity.

(D) Purification of Neutro~hil Inhibitorv Factor from Pichia Following methanol induction for 48 hours, 75 ml of Pichia cell supernatant (pHIL7SP-Nlc10) 48 hours following methanol induction was obtained by centrifugation for 15 minutes at 1,800 x gm~ and filtered through a 0.22 ~m cellulose acetate membrane.
This was concentrated using an Amicon s~irred UF cell equipped with a 10,000 molecular weight cut-off membrane (YM10) and then diluted with water (about 10-fold). This diafiltration process was repeated until the WOg4/14973 PCT~S93/12626 conductivity was reduced from 45 mS to 1 mS. The final volume of the concentrate was 25 ml.
This concentrate was dialyzed at 4C for 6 hours against one liter of 0.05 M bis Tris-propane-HCl, pH 7.0 to adjust the pH to neutrality, and then against two changes of one liter of 0.001 M potassium phosphate, pH
7Ø
Fifteen ml of the dialyzed cell supernatant was loaded onto a 0.8 x 15 cm column of ceramic hydroxyapatite (Pentax, 2 ~m; American International Chemical, Inc., Natick, MA) equilibrated with 0.001 M
potassium phosphate, pH 7.0 at a flow rate of 0.4 ml/min (48 cm/hour). The column was washed with one column volume of 0.001 M potassium phosphate, pH 7.0 and then developed with a linear gradient from 0.001 to 0.050 M
potassium phosphate over 20 column volumes at a flow rate of 0.35 ml/min. Substantial neuLLo~hil inhibitory activity eluted at approximately 0.02 - 0.035 M
potassium phosphate in much the same fashion as observed for ne~L~opllil inhibitory factor isolated from Ancylostoma caninum (Example 2(D)).
Fractions exhibiting substantial ne~L~o~hil inhibitory activity (A~s~ssed using the neutrophil-plastic adhesion assay (Example l(C))) were combined and concentrated to about 3 ml using an Amicon centrifugal concentrator equipped with a 10,000 molecular weight cut-off membrane (CentriPrep 10, Amicon, Beverly, MA) and applied to a 1 x 25 cm C4 300 A
reverse phase column (5 ~m particle size, Vydac, Hesperia, CA) equilibrated with 0.1% trifluoroacetic acid. The column was washed with four column volumes of equilibration buffer and then developed with a linear gradient of acetonitrile from 15 to 40%~over 10 column volumes at a flow rate of 5 ml/min. A major complex peak absorbing at 214, 254, and 280 nm eluted at about 36-38% acetonitrile.

W094/14973 PCT~S93/12626 `_ 95 ~152599 Fractions including and bracketing this peak were dried using a centrifugal evaporator to remove solvent and trifluoroacetic acid and rehydrated with 0.065 M
A potassium phosphate, pH 7.0, 0.08 M NaCl. The 5 rehydrated fractions ro~ceC~ substantial neutrophil inhibitory activity as judged by the neutrophil-plastic adhesion assay (Example l(C)) and the hydrogen peroxide release assay (Example l(E)).
Fractions with substantial activity were combined lO and sequenced by Edman degradation using a 47OA/12OA/9~OA gas phase seql~Pnc~r (ABI, Foster City, CA) (See Example 9) and yielded the following sequence:
Asn-Glu-His-Asn-Leu-Arg-Xxx-Pro-Gln-Xxx-Gly-Thr-Glu-Met-Pro-Gly-Phe-Xxx-Asp-Ser-Ile-Arg-Leu-Gln-Phe-Leu-15 Ala-Met-His-Asn-Gly-Tyr-Arg-Ser-Lys-Leu-Ala-Leu-Gly-His-Ile-Se r-Ile-Thr-Glu. "Xxx" refers to an undetermined amino acid at that position, since no specific amino acid was identified during Edman degradation of the peptide.
This sequence matches the predicted N-terminal sequence of NIF-lFL, the NIF isoform used in this construction construct (pHIL7SP-NlclO; see Figure 8).
The first position at which a residue was not detected is predicted to be a cysteine; cysteine residues could not be detected in this analysis because the protein had not been alkylated. The two other positions at which residues were not detected correspond to asparagine residues followed by either a serine or threonine one residue distant. This is a glycosylation consensus sequence tAsn-Xxx-(Ser/Thr)~ and the fact that asparagine was not detected ~LLO1IY1Y suggests that these asparagines are glycosylated. The C4-purified preparation was estimated to have an I ~ of about 5-lO
nM in the hydrogen peroxide release assay (Example l(E))-W094/14973 PCT~S93112626 2~S~S9 9 ExamPle 13Determination of SDecificitY of the Neutro~hil Inhibitory Factor To test the specificity of the Neutrophil Inhibitory Factor of the present invention, and to confirm that it did not inhibit neu~L~phil activation by a general cytotoxic mechA~;sm, the activity of the inhibitor was assessed in a non-neuLLu~hil cell adhesion-based assay, platelet aggregation.
The effects of the hookworm Neutrophil Inhibitory Factor on blood platelet aggregation were examined.
Platelet aggregation was performed with human platelet-rich plasma (PRP). PRP was stirred at 37C in an aggregometer (Scienco Model 247, Morrison, C0) and aggregation was initiated by the addition of lO ~M ADP
(Sigma, St. Louis, M0). AyyLeydtion was monitored as a change in light transmittance, and is expressed as the initial rate of a~y-eyation. A concentration of Neutrophil Inhibitory Factor of approximately 150 nM, a concentration that completely blocked neutrophil function as assessed by ne~L~hil-H W EC and neutrophil-plastic adhesion assays, homotypic neutrophil aggregation and hyd~G~en peroxide release by neutrophils, had no inhibitory effect on ADP-induced aggregation of human platelets.

Exam~le 14 CDllb/CDl8 Inteqrin is a Primary Rece~tor for Neu~LG~hil InhibitorY Factor from Hookworm (A) Immuno~reci~tation of 1~I-Labelled NIF Using Monoclonal Antibodies to CDllb/CDl8 in the Presence of Neutrophil Extract.
NIF purified from AncYlostoma cani~um was radiolabeled using the following method. Approximately 30 ~g NIF was labeled with 2 mCi Na125I (carrier free;
Amersham, Arlington Hills, IL) using Enzymobeads W094/14973 PCT~S93112626 `~ 2152599 (BioRad, Hercules, CA) Briefly, to a 1.5 ml eppendorf test tube was added 360 ~1 of the Enzymobead suspension together with 180 ~1 of a 1% beta-D-glucose solution, NIF and Nal~I. This mixture was allowed to react at room temperature for 30 minutes. Labeled NIF was separated from unbound 125I-iodine by desalting on a PD10-DG column (BioRad, Hercules, CA) using phosphate buffered saline (0.1 M sodium phosphate pH 7.2, 0.15 M sodium chloride) containing 1% bovine serum albumin as elution buffer.
Radioactive fractions contA;n;ng NIF were pooled. The specific act~vity of the t~I-NIF was 13.9 ~Ci/~g.
Various leukocyte proteins were ~Cpcce~ for ability to capture NIF in immunoprecipitation experiments. Potential cellular receptors for NIF were selected from a dete~ye~,L extract of leukocytes using specific monoclonal ant; hoA; es.
Leukocytes were prepared from human blood using Mono-poly (ICN, Biomedicals Inc., Costa Mesa, CA). The leukocyte cell pellet was resl~spenA~A in 1 ml resuspension buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl2) followed by the addition of 1 ml extraction buffer (2% Triton X-100, 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl2). Cells were ;n~l~hAted on ice 30-60 minutes, vort~Y; ng briefly every 10 minutes. Cell debris was pelleted at 5000 g for 5 minutes at 4C.
Monoclonal antibody-test protein complexes were formed by incubating 10 ~g specific monoclonal antibody with 200 ~l of leukocyte detergent extract at 4C for 4 hours. To this mixture was added 2.5 ~1 of the I~I-NIF
and these reagents were incubated at 4C for 18 hours.
Precipitation of the complex was effected by adding this mixture to a 1.5 ml eppendorf test tube containing 50 ~l of protein G-sepharose (Pharmacia, Pist~caway NJ;
resuspended in TACTS 20 buffer (0.05% Tween 20, 20 mM
Tris pH 8, 120 mM NaCl, 2 mM CaCl2) with 1% bovine serum albumin) and gently agitating at 4C for 2 hours.

WO94114973 PCT~S93/12626 The protein G-cPphArose beads were subsequently washed four times with TACTS 20 buffer. Fifty microliters of Laemmli sample buffer (Laemmli, U.K., 1970, Nature, 227:680-685) contAin;~g 5%
~-mercaptoethanol was then added to the aspirated beads;
this material was ;nc~lh~ted at 100C for 10 minutes and loaded onto 4-12% gradient SDS-polyacrylamide gels (Novex, San Diego, CA). Gels were dried after running and visualized by ex~G~e to X-Omat film (Kodak, Rochester, NY) in the presence Quanta III screens (Dupont, Wi~ington, DE) at -70C. Size stAnAArds were 4C-RA; nhow markers (Amersham, Arlington Hills, IL).
When monoclonal an~; ho~; es (MAb) directed to the CDllb/CD18 integrin complex (OKM-l, ATCC# CRL8026; LM-2, ATCC# HB204) were used in these experiments, I~I-NIF was precipitated as evi~nce~ by a band that migrated with an apparent mol~c~ r weight of approximately 41,000 daltons upon autoradiography. Precipitation of I~I-NIF
was dependent on the prPRPnCP of these ant; ho~; es as well as the prPcenr~ of leukocyte extract. Furthermore, the precipitation of I~I-NIF was not observed in the presence of a one hundred fold molar exsess of cold NIF.
~I-NIF did not precipitate when MAbs to other leukocyte integrins were used including MAbs directed against the VLA-4 (L25.3; Becton Dir-k;nsQn, Su~ vale, CA) and CDllc/CD18 (SHCL-3; Becton Dirk;nRon, Sunnyvale, CA) integrin complexes. A relatively minor amount of I~I-NIF
was observed when a MAb directed against the CDlla/CD18 (TSl/22; ATCC# HB202) integrin complex was used. This was likely due to cross-reactivity of the anti-CDlla/CD18 antibody with the related integrin complex CDllb/CD18. These results demonstrate that CDllb/CD18 is a cell-surface receptor f~r Ancylostoma caninum NIF on leukocytes.

WO94/14973 PCT~S93/12626 -(B) Preciitation of ~ CDllb/CDl8 Usinq Biotinylated NIF
As another approach to identify NIF receptors on leukocytes, biotin-labeled NIF was used to precipitate NIF-associating proteins from a detergent extract of surface iodinated leukocytes.
NIF was biotinylated by conjugation to its carbohydrate moieties. Approximately 16 ~g of NIF
purified from hookworm (Ancylostoma caninum) lysates (hydroxyapatite eluate; see Example 2(D)) was oxidized with 50 mM N~IO4 in 1 ml 0.1 M sodium acetate, pH 5.5.
After 20 minutes at 4C the reaction was terminated with the addition of 100 ~l 165 mM glycerol. Oxidized NIF
was separated from other reaction products using a Microcon 10 conc~ntrator (Amicon, Beverly, MA), and diluted into 100 ~l 0.1 M sodium acetate, pH 5.5.
Biotinylation was effected by the addition of 400 ~l 6.25 mM biotin-LC-hydrazide (Pierce, Skokie, IL). The reaction was allowed to proceed for 18 hours at 4C.
Biotinylated NIF was worked up by buffer exchange into phosphate buffered saline (PBS; 0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2), using a Microcon 10 concentrator. To 250 ~l of the concentrate was added an equal volume of glycerol, giving a final NIF-biotin concentration of approximately 32 ~g/ml. This material was stored at -20C.
The anti-CD18 integrin complex monoclonal antibodies LM-2 and OKM-1 (anti-CDllb/CD18; ATCC #HB204 and CRL8026, respectively) and TSl/22 (anti-CDlla/CD18;
ATCC# HB202) were biotinylated using the protocol described above.
Cell surface iodination of human leukocytes was done using the following ~oced~e. A ~otal leukocyte fraction, prepared from 90 ml of fresh human blood using Mono-Poly density gradient separation (ICN Biomedical, Costa Mesa, CA), was suspended in 0.5 ml phosphate WO94/14973 PCT~S93tl2626 ?,J~5?~5g9 loo buffered saline. To the cell sllspencion was added 2 mCi Nal~I (carrier free; Amersham; Arlington Heights, IL), 60 - ~l 0.03% hydrogen peroxide and 100 ~l lactoperoxidase at 2 mg/ml (BioRad; Hercules, CA). The reaction was allowed to proceed for 30 minutes at room temperature, with gentle agitation every two minutes. The reaction was terminated by the addition of 25 mM KI in PBS, and the cells were washed two times with PBS. The leukocyte cell pellet was resusp~n~A in 1 ml resl~pe~cion buffer and leukocyte extract was prepared as described above in Example 14(A~.
Sixty microliters of NIF-biotin (32 ~g/ml) was diluted with 40 ~l resuspension buffer and inc~hAted with 200 ~l ~I-labeled leukocyte extract at room temperature for 6 hours. Precipitation of NIF-associating proteins from the leukocyte extract was effected by the addition of 100 ~l ~L~e~Lavidin-agarose (Pharmacia; Piscataway, NJ) to this mixture. Test tubes were agitated gently for 18 hours at 4C. Beads were subsequently washed four times with 500 ~l TACTS-20 buffer (0.05% Tween 20, 20 mM Tris pH 8, 120 mM NaCl, 2 mM CaCl2), and associated proteins were solubilized with 50 ~l sample buffer (5% ~-mercaptoethanol) and analyzed by SDS-PAGE as described in Example 5. CG11 L~ ol precipitations were performed in a similar manner with biotinylated monoclonal anti hoA ies to CDllb/CD18 and CDlla/CD18.
Biotinylated NIF precipitated two I~I-labeled polypeptides that, when separated by 6% SDS-PAGE, had apparent molecular weights of about 170 kDa and about 95 kDa. These polypeptides comigrated on SDS-PAGE in this experiment with the two polypeptides that were precipitated by the anti-CDllb/CD18 mon~clonal antibodies LM-2 and OKM-l. This data strongly suggests that CDllb/CD18 is a major receptor for NIF on leukocytes when considered with the results of the WO94/14973 PCT~S93112626 101 215259~

previous experiment (Example 14(A)), in which CDllb/CD18 was shown to associate with NIF.

Example 15 Pre~aration Of Native Neutro~hil InhibitorY Factor From Toxocara canis (A) Pre~aration of Toxocara Lysate.
Frozen cAnin~ worms Toxocara canis were obtA;n~
from Antibody Systems (Bedford, TX) and were stored at -70C until homogenized. Toxocara canis were homogenized~on ice in homogenization buffer ~0.02 M
Tris-HCl pH 7.4, 0.05 M NaCl, 0.001 M MgCl2, 0.001 M
CaCl2, 1.0 X 1~5M E-64 Protease Inhibitor (CAS
66701-25-5), 1.0 X 10 M pepstatin A
(isovaleryl-Val-Val-4-amino-3-hydroxy-6-methyl-heptanoyl -Ala-4-amino-3-hydroxy-6-methylheptanoic acid, CAS
26305-03-3), 1.0 X 10-5M chymostatin (CAS 9076-44-2), 2.0 X 105M APMSF (amidinophenylmethylsulfonyl fluoride-HCl), 5% (v/v) glycerol] using an Ultra-Tarrax homogenizer (Janke and Kunkel, Stanfen, Germany). The protease inhibitors E64, pepstatin A, chymostatin, and APMSF`were obtained from Calbiochem (La Jolla, CA). Approximately 3-6 ml of homogenization buffer was used to homogenize each gram of frozen worm. Twenty-four grams of worms was used in total. Insoluble material was pelleted by two se~quential centrifugation steps: 40,000 X gm~ at 4OC
for 25 minutes followed by 105,000 X gmU at 4C for 1 hour. The supernatant solution was clarified by passage through glass wool and a 0.45 ~m cellulose acetate filter (CoStar, Cambridge, MA).

(B) Concanavalin A Se~harose Chromatoqra~hy of Toxocara LYsate Toxocara canis lysate (68 ml) was absorbed to 26 ml of Concanavalin A Sepharose (Pharmacia, Piscataway, NJ) pre-equilibrated with Con A buffer tO.02 M Tris-HCl, pH

W094/14973 PCT~S93112626 ~5~ 102 7.4, l M NaCl, O.OOl M CaCl2, O.OOl M MnSO4] by recycling it through a l.6 X 13 cm column at a flow rate of 4 ml/minute (ll9 cm/hour) for 2 hours. The column was at room temperature (24C) while the reservoir of lysate was maintained on ice throughout the procedure. The column was subsequently washed with lO0 ml of Con A
buffer. Material that had activity in anti-adhesion assays (see, Section (D) below) was eluted with approximately 3-5 column volumes of Con A buffer containing 0.5 M methyl-alpha-mannopyranoside (CAS
617-04-09) at a flow rate of l ml/minute (30 cm/hour).
The eluted material was concentrated to 5 ml using an Amicon stirred ultrafiltration vessel equipped with a lO,OOo molecular weight cutoff membrane, then diluted to 50 ml with deionized water, and reconcentrated to 2.3 ml using a centrifugal ultrafiltration unit with a lO,000 molecular weight cut-off (Polysciences, Inc., Warrington, PA) Material used for molecular sieve chromotography with Su~eldex columns (1.5 ml) was additionally concentrated to 0.5 ml using centrifugal ultrafiltration units with a lO,000 molecular weight cut-off (Amicon, Inc., Beverly, MA).

(C) Molecular Sieve Chromatoqra~hy Usinq Su~erdex 200 HR.
Material eluted from immobilized Concanavalin A
(see step (B) above) and concentrated by ultrafiltration was loaded on a l.0 cm X 30 cm column of Superdex 200 HR
(Pharmacia, Piscataway, NJ). The column was pre-equilibrated with O.Ol M potassium phosphate, pH
7.35, and 0.15 M NaCl at 24C. The chromatography was conducted at a flow rate of 0.25 ml/minute.
Anti-adhesion activity eluted with an a~parent molecular weight of approximateiy 20,000.

WO94/14973 PCT~S93tl2626 2l5259g (D) AssaY of Neutrophil InhibitorY ActivitY Isolated From Toxocara canis Material eluted from Concanavalin A SDphArose with ; methyl alpha-mannopyranoside was assayed by the neutrophil-HUVEC adhesion assay (see Example l(B)) and was found to inhibit the adhesion of neuL~ophils to endothelial cells. Adhesion inhibitory activity was also demonstrated using the neuLL~hil-plastic adhesion assay. (Example l(C)).
Material purified by chromatography on both Concanavali~ A SDphArose and Superdex 200 HR inhibited neutrophil adhesion in the neutrophil-adhesion assay (see Example l(C)).

Examle 16 In Vivo Characterization Of Neutrophil InhibitorY Factor Neutrophil Inhibitory Factor isolated from canine hookworms was tested in an animal model of acute inflammation.
Peritoneal inflammation was induced in 150-250 gram Sprague-Dawley rats by an intraperitoneal injection of nine ml of 2% oyster glycogen in ~O (see Baron et al., Journal of Immunoloaical Methods, 49:305, 1982; McCarron et al., Methods in Enzymoloqy, 108:274, 1984; Feldman et al., Journal of Immunoloa,,Y, 113:329, 1974; Rodrick et al., Inflammation, 6:1, 1982; and Kikkawa et al., LaboratorY Investiqation, 30:76, 1974).
NIF was prepared as described in Example 2. Lysate from approximately 20,000 hookworms (48.2 g wet weight) was prepared and chromatographed on ConA, Superdex, and hydroxyapatite (HA). The active fractions from two equivalent HA runs were combined to yield 41 ml of HA
material. One ml of NIF solution (11 ~ was administered simultaneously with the glycogen by the intraperitoneal route or thirty minutes prior to 3~ glycogen administration by the intravenous route. Four W094/14973 PCT~S93/12626 ~5~ 104 hours later the peritoneal exudate was harvested by purging the peritoneàl cavity with 30 ml of Hanks Balanced Salt Solution without Ca++ or Mg++, supplemented with 0.03% EDTA and blood cells were counted on a Celldyn 3000 (Abbott Laboratories, North Chicago, IL) automated multiparameter differential cell counting instrument. The major cellular component in the exudate was neuLlo~hils. Figure lO depicts the effects of varying doses of NeuL~Gphil Inhibitory Factor isolated from canine hookworms on neu~ophil infiltration in peritoneal ~nflammation in rats induced by interperitoneal infusion with glycogen. Glycogen (9 ml) and Neutrophil Inhibitory Factor (l ml) were injected simultaneously by intraperitoneal route.
Figure lO shows the results of six independent experiments. NIF caused a dose ~epe~nt inhibition of neutrophil infiltration to the rat peritoneal cavity in response to glycogen.
A second study was performed to determine if intravenous administration of NIF could prevent glycogen-induced rat peritoneAl inflammation. In one set of rats, NIF and glycogen were administered by the intraperitoneal route as previously described. In a second group of rats, l ~g of NIF was administered intravenously thirty minutes prior to the intraperitoneal infusion of glycogen. A third group of animals received glycogen and NIF treatment was replaced with saline. Four hours later the peritoneal exudate was collected and blood cells were counted.
Figure ll depicts the effect of Neutrophil Inhibitory Factor isolated from canine hookworms on neutrophil infiltration in peritoneal inflammation in rats induced by intraperitoneal infusio~ of glycogen.
Neutrophil Inhibitory Factor (l ml) was injected by intraperitoneal route in conjunction with intraperitoneal infusion of glycogen, or by intravenous WO94/14973 PCT~S93/12626 105 21 ~2 599 route thirty minutes prior to infusion of glycogen.
Figure 11 represents a summary of the six independent experi~ents for the intraperitoneal administration of NIF and the results of the single experiment for the intravenous administration of NIF. These results demonstrate that NIF, when administered by either the intraperitoneal or intravenous route, was effective in the prevention of peritoneal inflammatory response in glycogen-stimulated rats.

Example 17 ~
Inhibition of NeuL~G~il-Mediated Inflammation In Vivo bY Recombinant Neutrophil InhibitorY Factor The in vivo anti-inflammatory properties of recombinant NIF (rNIF) were tested in a rat ear inflammation assay (adapted from Young et al., 1984).
In this assay, inflammation was in~llre~ in the rat ear by topical administration of arachidonic acid.
Sprague-Dawley rats (250g) were anesthetized with pentobarbital (initial dose of 65 mg/kg intraperitoneal;
Anpro Pharmaceutical, Arcadia, CA); rats were maintained at a surgical plane of anesthesia for the-duration of the experiment (4 hours). A catheter was inserted into the femoral vein of the anesthetized rat. One hundred microliters of recombinant NIF (produced in Pichia pastoris; see Example 12) at a concentration of 20 mg/ml in PBS was injected via the catheter. Co,.L~ol rats received 100 ~L sterile 0.14 M NaCl. Five minutes after the IV administration of rNIF, arachidonic acid (Sigma, St. Louis, MS; diluted 1:1 with acetone to a final concentration of 500 mg/ml) was applied to the right ear in three 10 ~1 applications each to the i~side and the outside of the ear. The right ear thus~eceived a total dose of 30 mg arachidonic acid. The left ear, used as a background control, received a total of 60 ~1 acetone.

WO94/1497~ PCT~S93/12626 Four hours after administration of arachidonic acid the rat was sacrificed with CO2.
Neutrophil infiltration into the arachidonic acid-treated ear tissue was quantitated indirectly by determining myeloperoxidase activity. A tissue sample was obtained from the center of each ear using a 7 mm skin punch tMiltex; Lake Success, NY). The tissue sample was cut into small pieces and added to a 16 x 100 mm test tube that contained 0.5 ml HTAB buffer (0.5%
hexadecyltrimethylammonium bromide in 50 mM sodium phosphate, pH 6.4; HTAB was purch~se~ from Sigma, St.
Louis, MO). The ear tissue was homogenized for 20 seconds using an Ultra-Turrax (Janke and Kunkel;
Staufen, Germany) at high speed. Insoluble matter was removed from the homogenate by centrifugation at 14,000 x g for 10 minutes followed by filtration through Nytex gauze. Myeloperoxidase determinations were done in triplicate in 96 well poly~y~el.e plates (Costar;
Cambridge, MA). Twenty five microliters of HTAB-solubilized ear tissue was added to each well, and to this was added 100 ~l of substrate solution.
Substrate solution comprised two components: (1) 0.012%
H2O2 in 0.1 M sodium acetate pH 4.5 and (2) 0.3 mg/ml 3,3',5,5'-tetramethylbenzidine in 10% HCl, combined immediately prior to use at a ratio of 0.125:1. After ten minutes the reaction was stopped by the addition of 125 ~l 1 M H2SO4. Samples were quantitated colorimetrically at 450 nm and background was read at 650 nm. A stA~Ard curve was generated using human leukocyte myeloperoxidase (Sigma; St. Louis, M0).
Recombinant NIF had a protective effect on arachidonic acid-induced neutrophil infiltration into ear tissue. Figure 12 shows that ear t~ssue from rats that received rNIF had a mean of 1.6 myeloperoxidase units/ml (MU/ml) whereas ears from rats that received saline had a mean of 4.1 MU/ml, when background WO94/14973 PCT~S93/12626 myeloperoxidase activity is subtracted (n=10 in each group). One myeloperoxidase unit will produce an increase in absorbance at 470 nm of 1.0 per minute at pH
7.0 and 25C, calculated from the initial rate of reaction using guaiacol as substrate (Desser, R.K., et al., Arch. Biochem, Biophys. 148:452 (1972)).
Neutrophil infiltration was thus r~ se~ -60% in rats that received rNIF (8 mg/kg IV); there is a significant difference at the 95% confidence level between rats that received NIF and rats that received saline (Student's t test). These results are consistent with the demonstration that hookworm-derived NIF prevented neutrophil infiltration into the peritoneal cavity of rats in response to glycogen (see Example 16). These data further provide evidence that rNIF acts as a potent anti-inflammatory agent n vivo.

Example 18 The Use of Neutrophil Inhibitorv Factor DNA Seauences to Isolate Neutrophil Inhibitorv Factor-Related Proteins NIF cDNA se~lPnc s are used as probes to isolate DNA sequences that encode proteins that are functionally and structurally related to NIF.
Genomic DNA or cDNA libraries are formed using standard procedure (for example see Molecular Cloning.
A Laboratory Manual. Sambrook, J., Fritsch, EF., and Maniatis, T. 2nd Ed. Cold Spring Harbor Laboratory Press, CSH, NY 1989). These libraries may be from any animal, fungal, bacterial or viral source, such as AncYlostoma caninum, other Ancylostoma species, other helminths and mammals including human placental tissue.
Such libraries are screened for useful clones by nucleic acid hybridization using NIF cDNA sequences isolated from Ancylostoma as probe. For example, NIF
cDNA fragments of about 100-2000 base pairs labeled for detection by standard procedure (for example, see W094/14973 PCT~S93112626 99 ~

Molecular Cloning. A Laboratory Manual. Sambrook, J., Fritsch, EF., and Maniatis, T. 2nd Ed. Cold Spring Harbor Laboratory Press, CSH, NY 1989) is hybridized with a library from another tissue or another species under conditions of variable stringency. More preferably, however, reduced stringency hybridization conditions are utilized (eg 6X SSC tSSC is 150 mM NaCl, 15 mM trisodium citrate], 0.02 M sodium phosphate pH
6.5, 5X Denhardt's solution, 0.5% (w/v) SDS, 0.01 M
lo EDTA, 100 ~g/ml sheared, denatured salmon sperm DNA, O.23% dextr~n sulfate, 20-30% formamide at 42C for 18 hours). Also, more preferably, re~lce~ stringency conditions are used to wash filters after hybridization (0.5 to 2X SSC at 45-60C for 20 minutes after two prewashes with 2X SSC for 15 minutes).
NIF-related complementary DNAs isolated using the t~rh~i ques described above are subjected to nucleotide sequence analysis using the procedure of dideoxy sequencing (Sanger et al, 1977, Proc. Natl. Acad. Sci.
USA 74:5463-5467). Isolates containing open reading frames (i.e., initiating with a methionine and terminating with a TAA, TGA or TAG stop codon) are inserted into suitable vectors for protein expression in either bacterial, yeast, insect or mammalian cells.
Expression systems comprise vectors designed to secrete recombinant protein (i.e., fusion of cDNA isolate open reading frame with a known secretion signal sequence for that cell type) into the culture medium. Vectors lacking a homologous secretion signal sequence are also used for expression. Either conditioned media or cell lysate, depending on the expression system used, is tested for inhibitory activity using one or more of the following criteria for neutrophil activ~tion: release of hydrogen peroxide, release of superoxide anion, release of myeloperoxidase, release of elastase, homotypic neutrophil aggregation, adhesion to plastic surfaces, WO94/14973 PCT~S93112626 ~_ 21525~9 adhesion to vascular endothelial cells, chemotaxis, transmigration across a monolayer of endothelial cells and phagocytosis.
Proteins that are structurally related to NIF and that are inhibitory in one or more of these neutrophil function assays would be considered to belong to the NIF
family of related molecules.

Example 19 Expression of Functional Recombinant NIF in E. coli DNA for the NIF-lFL coding region, initiating at the codon that corresponds to the N-terminal methionine, is inserted into an E. coli expression vector. Examples of such vectors are given in R~lhAs, P. and Bolivar, F., 1990 (Methods in Enzymology, 185:14-37). The DNA is inserted into the E. coli expression vector using methods similar to the methods of insertion of the NIF-lFL coding region into mammalian and yeast expression vectors described in Examples 11 and 12, respectively. PCR oligonucleotide primers are designed to generate an amplification product that contains the NIF-lFL coding region. As was described in connection for the methods for insertion of NIF-lFL into mammalian and yeast expression vectors (see Examples 11 and 12, respectively), primers are engineered so that this fragment contains 5' and 3' restriction sites that are compatible with insertion into the selected expression vector. The expression construct is preferably engineered so that the recombinant NIF will be secreted into the cytoplasm and not the periplasmic space. This may be accomplished by omitting an E. coli secretion - signal from the construct.
E. coli cells are transformed with~the NIF-lFL
expression vector construct using standard methods.
(See, e.a., Molecular Cloning A Laboratory Manual, Sambrook, J. Fritsch, E.F. and Maniatis, T., Second W094114973 PCT~S93/12626 599 llo Edition, Cold Spring Harbor Laboratory Press, 1989, 1.74-1.84). Cells are grown in a~LGpLiate media (e.q.
Luria Broth; see Molec~llAr Cloning. A Laboratory Manual, Sambrook, J. Fritsch, E.F. and Maniatis, T., Second Edition, Cold Spring Harbor Laboratory Press, 1989, A.1) and harvested before they reach the stationary phase of growth.
The majority of the recombinant NIF should be present in the cytoplasm in the form of insoluble and lo functionally inactive aggregates. The solubilization and refolding of the recombinant protein present in these aggregates may be accomplished using known methods such as those reviewed in detail in Kohno et al., 1990 (Methods in EnzymologY, 185:187-195). Refolded recombinant NIF may be separated from unfolded recombinant NIF and other reaction products using a number of stAnA~rd chromatographic teçhniques, including C4 reverse phase HPLC (see, e.a., Example 2(E)).
Refolded recombinant NIF is tested for functional activity using the nehL~o~hil function assays described in Example 1.
This recombinant NIF is not glycosylated.

Exam~le 20 Pre~aration of Functional Recombinant NIF bY Refoldinq 'Insoluble' MethionYl-NIF Produced in the E. coli Cytoplasm (A) Description of the E. coli expression vector pMa5-NI1/3 PCR oligonucleotide primers were designed to generate an amplification product that contains the NIF-lFL coding region. The PCR product initiates at the first Asn-codon of mature NIF-lFL which~as a result of the amplification was changed from AAT to AAC. The 3'-primer replaces the TGA translational stop codon by a TAA triplet and introduces a SpeI, a HindIII and a BglII

WO94/14973 PCT~S93112626 ~ 21 ~2599 111 :

site downstream-of the coding region. The PCR primers were as follows:

Pst414:
5'-CCTCCTCCTA-GATCTAAGCT-TACTAGTTTA-TAA~l~lCGG-AATC~.~T~
A-ACTC (54-mer; 3'-primer matching with the C-terminus) Pst415:
5'-AACGAACACA-ACCTGAGGTG-CCCG (24-mer; 5'-primer matching with the N-terminus) After digestion with HindIII, the correctly sized PCR fragment was isolated from agarose-gel and inserted on an E. coli expression vector downstream of the phage lambda PR promoter. The recipient vector was opened with NcoI, treated with DNA polymerase I (Klenow fragment) and subsequently digested with HindIII. The resultant vector, designated pMa5-NIl/3, is schematically shown in Figure 14; the seguence of the relevant part of the vector is shown in Figure 15. The NIF-lFL region present in this vector was entirely se~Pnc~ to rule out the presence of unwanted mutations. The expression module consists of the following elements: (1) The phage lambda PR promoter. (2) A small cistron which is present upstream of the Met-NIF-lFL region; this upstream cistron includes the first nine codons of the phage lambda cro gene and terminates at the TAA stop located in between the Shine Delgarno (SD)-box and the ATG
initiator codon of Met-NIF-lFL (see Figure 14 and 15).
~ The leader-cistron has the potential to code for a 31 residue polypeptide. Such a two-cistron arrangement, in addition to being found in a number of 'natural' operons, has been used succesfully for ~mproving the expression of heterologous genes whose level of expression is thought to be limited by the initiation of translation (Schoner et al., PNAS 81:5403-5407 (1984);

Wog4/14973 PCT~S93/12626 ~ ~ 112 Spanjaard et al., Gene 80:345-351 (1989); Makoff and Smallwood, NAR 18:1711-1718 (1990). (3) An open reading frame encoding Methionyl-NIF-lFL. The construction scheme is indeed such that the NIF-FL1 5'-end is correctly fused to an ATG initiator codon. Upstream of this ATG codon a SD-sequence (eg, GGAGGT; see Figures 14 and 15) is present. (4) Two copies of a phage fd derived transcription terminator (fdT) downstream of the Met-NIF-lFL coding region.

(B) Produc~ion of 'insoluble' methionyl-NIF.
To assess the effectiveness of pMaS-NIl/3 in expressing the NIF-lFL gene, the vector was introduced in W3110 cells harboring pcI857. The plasmid pcI857 specifies resistance to kanamycin (20 ~g/ml), encodes a temperature sensitive repressor of the lambda PR
promoter, and is compatible with the pMa5-NI1/3.
Cultures were grown in LB medium at 28-C to a density of about 2 x 108 cells/ml and then induced at 42-C for 2-3 hours. Analysis of total cellular extracts of induced and non-induced cells by SDS-PAGE indicated that a new -33 kDa protein (calculated molecular weight of NIF-lFL
= -29 kDa) is synthesized upon thermo-induction of the promoter. In addition to total cellular extracts, we also analysed the pellet (insoluble) and supernatant (soluble) fraction obtained by opening the induced cells by sonication and clearing the lysate by centrifugation.
The results indicated that the newly synthesized -33 kDa protein precipitates intracellularly, i.e. forms so-called inclusion bodies. Following fractionation on an SDS-polyacrylamide gel, transfer onto ProBlott (ABI) and visualization by coomassie-staining, the -33 kDa band was excised and its N-terminal amin~ acid sequence determined. The sequence obtained was: M-N-E-H... .
This result demonstrated that the initiator methionine was not removed from the primary translation product and WO94/14973 PCT~S93/12626 -2I52~99 clearly identified the 33 kDa band as recombinant NIF.
; It was estimated that the recombinant NIF protein accumulates to -10 mg per liter and per OD~o unit.
;

(C) Renaturation of NIF ~rotein expressed in E. coli.
W3110 E. coli cells cont~ining the plasmid pMa5-NIl/3 were grown in a shake flask incubator in six liter flasks each contAining 1.5 liters of LB media at 28 C until the optical density (OD) was in the range of 0.6-0.9 au at 550 nm. An additional 1.5 liters of LB
media at 56~C was added to each flask to induce expression of recombinant NIF, and the flasks were incubated at 42 C with shA~ing. The OD was monitored and cells were harvested by centrifugation when the OD
was within the range of 1.0-1.5. The cell pellets were frozen at -80 C. Each tube contAin~ about 3.5 g cells.
Fifteen milliliters of TES buffer (0.05 M Tris, 0.05 M sodium ethylen~;Aminetetraacetate, 15% (w/v) sucrose, pH 8.0) was added to one tube, and the tube was sonicated to thaw and disperse the cell pellet. The suspension was then distributed into two 30 ml glass centrifuge tubes. An additional 2.5 ml of TES was used to wash the original tube and this wash solution was added to the glass tube. The suspensions in the glass tubes were sonicated (Branson Sonic Power Co., Danbury, Connecticut) four times for 30 seconds each, with an ice incubation between sonications to maintain the temperature S10 C throughout the procedure. The tubes were then centrifuged at 10,000 rpm for 20 minutes (12,100 x gm~) at 4-C. The supernatants were discarded.
The pellets were resuspended in 15 ml of PSX buffer (0.02 M potassium phosphate, 1 M sodium chloride, 1~
(v/v) Triton X-100, pH 7.2) per tube an~ sonicated at a low setting to break up the pellets followed by a 15 second sonication at medium power. The tubes were cooled on ice, resonicated at medium power for 15 W094/14973 PCT~S93/12626 $99 - ` 114 seconds, and then centrifuged at 10,000 rpm for 20 minutes (12,100 x gm~). The supernatant was ~;sc~rded.
The entire PSX resuspension/centrifugation process was repeated two additional times.
The pellets were resusp~n~P~ in 15 ml of PBS buffer (0.01 M sodium phosphate, 0.15 M sodium chloride, pH
7.3) per tube, briefly sonicated, and centrifuged as before. The PBS resuspension was repeated one additional time.
The pellets were then resuspended in PBS by sonication, 12.5 ml per tube. The contents of both tubes were combined, and the volume was brought to 30 ml by the addition of further PBS. The protein concentration of the purified inclusion bodies was determined using the DC Protein Assay (Bio-Rad, Hercules, California).
An aliquot of purified inclusion bodies contA;n;ng 5 mg of protein was placed in each of several 1.7 ml plastic microcentrifuge tubes. The tubes were microcentrifuged for 10 minutes at 4 C. The supernatants were ~ c~rded. Each pellet was resuspended in 1 ml of 0.05 M Tris, 1 % (w/v) Sarkosyl, pH 7.5 using sonication.
The tubes were vortexed at 37 C for 48 hours using a Thermomixer vortexer (Eppendorf, Hamburg, Germany).
Following this incubation, samples were submitted for NIF activity assays (see Example 1). Typically, activity corresponding to the activation (refolding) of 3% of the NIF present was found.

Example 21 Isolation and Characterization of NIF from AncYlostoma canlnum (A) Cloninq and Seauencina of NIF seauences from A.
caninum WO94/14973 PCT~S93/12626 -115 21 ~$2 59 9 Two new full-length coAi~g regions that code for r proteins related to NIF-lFL (see Example 10) were identified by PCR te~hnology using single stranded oligonucleotide DNA primers that match with the NIF-lFL
5 N- and C-terminal ends. These primers were as follows:

YGl:
5'-ATG-GAG-GCC-TAT-CTT-GTG-GTC-TTA (5'-primer matching with the N-terminal region enco~; ng: M-E-A-Y-L-V-V-L) YG2: ~
10 5'-TCA-TAA-CTC-TCG-GAA-TCG-ATA-AAA-CTC 13'-primer matching with C-terminal sequence corresponding to:
E-F-Y-R-F-R-E-L-stop codon) The YGl/YG2 primer couple was found to yield a correctly sized PCR product when using a total RNA
15 preparation of A. caninum (see Example 10) as template.
First strand cDNA synthesis (First-Strand cDNA Synthesis Kit of Pharmacia, uprcAl A ~ Sweden; 10 pmoles of the YG2 primer; -15 ~g of total RNA) and the subsequent amplification by PCR were carried out according to the 20 manufacturer's specifications. The PCR was carried out with Taq DNA polymerase (Boehringer, Mannheim, Germany), 100 pmoles of both YGl and YG2 and using 30 temperature cycles (1 minute denaturation step at 95 C; 1 minute annealing period at 55 C; 1.5 minute elongation step).
25 The obtained PCR product was isolated from agarose gel and cloned onto a phagemid vector (allowing preparation of single stranded DNA). Three clones, designated PCR-NIF5, PCR-NIF7 and PCR-NIF20, were retained for sequence determination. PCR-NIF5 was found to be 30 identical to NIF-lFL. PCR-NIF7 and PCR~Nlr20, however, represent two new NIF sequences. Their sequences are shown in Figure 16.-WOg4/14973 PCT~S93/12626 ` 116 Additional full-length A. caninum NIF sequences were isolated by screening a cDNA library using as hybridization probe a radiolabeled PCR fragment obtained with primers that target se~nC~c which are well conserved among the seven A. caninum NIF sequences described in Example 10 (lFL, 3P, 2FL, 3FL, 4FL, 6FL and lP). The following primers were used:

YG3:
5'-CAC-AAT-GGT-TAC-AGA-TCG-AGA-CTT-GCG-CTA-GGT-CAC the 5'-primer t~rgeting the region which in NIF-lFL encodes the amino acid sequence H-N-G-Y-R-S-K-L-A-L-G-H) - YG4:
5'-T-TTT-TGG-GTA-GTG-GCA-GAC-TAC-ATG the 3'-primer targeting the region which in NIF-lFL ncoAes H-V-V-C-H-Y-P-K-(I) Poly(A+) RNA was prepared from adult worms using the QuickPrep mRNA Purification Kit (Pharmacia, Uppsala, Sweden). Using this poly(A+) RNA preparation as template, an amplification product of about the expected length was obtained with the YG3/YG4 primer couple (the PCR conditions were as descibed above). The amplification product was shown by gel-electrophoretic analysis to be rather heterogeneous with respect to length. The YG3/YG4 primers were ; nA~ designed to target sequences that flank that part of the coding region where the various NIF sequences display significant differences in length; the heterogeneous nature of the PCR product indicated that the primers are useful for the amplification of several different isoforms. The PCR product were gel-pur~fied and subsequently radiolabeled by "random primer extension"
(~QuickPrime Kit~; Pharmacia, Uppsala, Sweden) for use as hybridization probe. A cDNA library was constructed W094114973 PCT~S93/12626 using described procedures (Promega Protocols and Applications Guide 2nd Ed.; Promega Corp.). About 3 ~g of mRNA was reverse transcribed using an oligo(dT)-NotI
primer-adaptor [S'-TCGCGGCCGC(T)~5; Promega Corp., Madison, WI] and AMV (Avian Myeloblastosis Virus) reverse transcriptase (Boehringer, M~nnheim, Germany).
The enzymes used for double stranded cDNA synthesis were the following: E. coli DNA polymerase I and RNaseH from BRL Life Technologies (Gaithersburg, MD) and T4 DNA
polymerase from Pharmacia. The obtained cDNA was treated with EcoRI methylase (RiboClone EcoRI Linker Ligation System; Promega). The cDNAs were digested with NotI and EcoRI, size selected on a 1% agarose gel (fragments of between 1000-7000 base-pairs were eluted using the Geneclean protocol, BI0101 Inc., La Jolla, CA), and unidirectionally ligated into the EcoRI-NotI
arms of the lambda gtll Sfi-Not vector (Promega). After in vitro packaging (GigapackII-Gold, Stratagene, La Jolla, CA) recombinant phage were obtained by infecting strain Y1090 (Promega). The usef~lnecs of the cDNA
library was demonstrated by PCR analysis (Taq polymerase from Boehringer; 30 temperature cycles: 1 minute 95 C; 1 minute 50 C; 3 minutes 72-C) of a number of randomly picked clones using the lambda gtll primer #1218 (New England Biolabs, Beverly, MA) in combination with the above mentioned oligo(dT)-NotI primer adaptor. The majority of the clones was found to contain cDNA inserts of variable size.
Approximately 1x106 cDNA clones (duplicate plaque-lift filters were prepared using Hybond~-N;
Amersham, Buckinghamshire, England) were screened with the radiolabeled YG3/YG4 PCR fragment using the following prehybridization and hybridiz~tion conditions:
5X SSC (SSC: 150 mM NaCl, 15 mM trisodium citrate), 5X
Denhardt's solution, 0.5% SDS, 50% formamide, 100 ~g/ml sonicated fish sperm DNA (Boehringer), overnight at WO94114973 PCT~S93/12626 2152~99 118 42 C. The filters were washed 4 times in 2X SSC, 0.1%
SDS at 37-C~ After overnight exposure to X-ray film, t numerous plaques that hybridized to the probe were identified; it was estimated that about 0.1-0.2% of the 5 clones scored positive. After a second hybridization round (24 positives were analyzed at lower plaque-density so as to isolate single pure clones), a number of phage clones were subjected to PCR anlysis and those cDNA inserts which were found to be large enough 10 to encompass the entire coding region were subcloned as SfiI-NotI fragments on pGEM-type phagemids (Promega).
We have determined the sequence of eight of these NIF
cDNAs (i.e., AcaNIF3, AcaNIF4, AcaNIF6, AcaNIF7, AcaNIF9, AcaNIF18, AcaNIFl9, and AcaNIF24). The data 15 are shown in Figure 16.

(B) ExDression of Functional NIF Proteins from A.
caninum in Pichia Pastoris.
The segments of DNA encoAing AcaNIF24, AcaNIF6, AcaNIF4, and AcaNIF9 were PCR amplified using pGEM-type 20 vectors cont~;n;ng the respective cDNAs (see above) as template. The 5'-primers contained no restriction sites and matched with the 5'-end of that part of the coding regions corresponding to the mature protein. Also, the first codon was altered from AAT to AAC (both codons 25 translate to asparagine). The sequences of the 5'-primers for the various NIF sequences were as follows:

AcaNIF24: 5'-AAC-GAA-CAC-AAC-CTG-ACG-TGC-CC
AcaNIF6: 5'-AAC-GAA-CAC-AAA-CCG-ATG-TGC-CAG-C
30 AcaNIF4: 5'-AAC-GAA-CAC-AAA-CCG-ATG-TGC-GAG
AcaNIF9: 5'-AAC-GAA-CAC-GAC-CCA-ACG-TG~CC

The 3'-primers were composed of 8 codons at the 3'end of the coding region, a TAA stop replacing the TGA

W094/14973 PCT~S93112626 -119 21~2~99 stop of the natural gene, and three unique restriction endonuclease sites (SpeI, HindIII, and ~glII). The 3'-primers used were:
-AcaNIF24:
5'-CCT-CCT-CCT-AGA-TCT-AAG-CTT-ACT-AGT-TTA-AAA-TCG-ATA-A
AA-CTC-CTT-GCT-ATC
AcaNIF6:
5'-CCT-CCT-CCT-AGA-TCT-AAG-CTT-ACT-AGT-TTA-TAA-CTC-TCG-G
AA-TCG-ATA-AAA-CTC
AcaNIF4: ~
5'-CCT-CCT-CCT-AGA-TCT-AAG-CTT-ACT-AGT-TTA-TAA-CTC-TCG-G
AA-TCG-ATA-AAA-CTC
AcaNIF9:
S'-CCT-CCT-CCT-AGA-TCT-AAG-CTT-ACT-AGT-TTA-TAG-CTC-TCG-A
AA-CGG-ATA-AAA-ATA

Amplification was accomplished using lO0 pmoles of each primer, 2 units of Vent polymerase in lx Vent buffer (New England Biolabs, Beverly, MA), 0.2 mM of each dNTP and lO0 ng of template DNA. The PCR
conditions were the same for all twenty cycles:
denaturation at 95-C for l minute, primer annealing at 60-C for l minute, and amplification for l.5 minutes at 72 C. The amplification product was gel-purified and digested with SDeI. The amplification product was ligated into StuI-S~eI cleaved pHIL7SP8 using standard methods. The ligation mixture was used to transform E.
coli WK6 selecting for ampicillin resistant clones. In each case a correct clone was identified by restriction and DNA sequence analysis. These plasmids, designated pYAM7SP-AcaNIF24, pYAM7SP-AcaNIF6, pYAM7SP-AcaNIF4, and pYAM7SP-AcaNIF9, were used to transform~the P. ~astoris yeast strain GTSll5(his4), as described in Example 12(B). Selection of His+ transformants and subsequent selection for NIF expression were performed as described WO94/14973 PCT~S93112626 2~525~ 9 120 in Example 12(B). The accumulation of functional NIF
protein in Pichia cell supernatant was detected and quantified using the LM2/CDllb/CD18 based ELISA with 3D2-HRP detection (Example 1) in the case of AcaNIF24, AcaNIF6, and AcaNIF4 and using the competitive assay for LM2/CDllb/CD18 (Example 1) in the case of the AcaNIF9.

(C) Purification and Characterization of NIF proteins AcaNIF24. AcaNIF6. AcaNIF4. and AcaNIF9.
The functionally active recombinant AcaNIF24, AcaNIF6, an~ AcaNIF4 proteins were purified and characterized in greater detail. Following methanol induction for 48 hours, Pichia cell supernatants were obtained by centrifugation for 15 minutes at 1,800 x g.
In the case of the recombinant proteins AcaNIF24 and AcaNIF6, the 250 ml supernant was adjusted to pH 7.0 by adding Tris-HCl and kept overnight at 4 C.
Precipitated material was removed by centrifugation.
The cleared supernatant was loaded on a 3D2-immunoaffinity resin and bound material eluted with glycine-HCl pH 2.5 (Example 27). The eluted fractions were neutralized and concentrated by ultrafiltration.
Both proteins were found to migrate as a single band on SDS-PAGE (4-20~ gradient gel; Novex). Edman degradation confirmed that correctly processed proteins were produced. The following N-terminal amino acid sequences were found:

AcaNIF24: N-E-H-X-L-T-X-P-Q-N
AcaNIF6: N-E-H-K-P-M-X-Q-Q-X-E-T-E-M-P
where X represents an unidentified residue.

The concentrations were determined~spectrophoto-metrically and the samples were assayed in the plastic adhesion and peroxide release assays (see Example 1).
Both recombinant AcaNIF24 and AcaNIF6 proteins were W094/14973 PCT~S93/12626 found to be equally active as recombinant NIF-lFL
~ (Figure 17).
Recombinant AcaNIF4 was purified by hydroxyapatite - and reverse-phase chromatography essentially as described in Example 23, however, the gel filtration step on Superdex was omitted. The purified protein was found to migrate as a single band on SDS-PAGE (4-20%
gradient gel; Novex). The conoe~ ation was determined spectrophotometrically. The results obtained in a competitive binding assay with biotinylated NIF-lFL
indicated that AcaNIF4 had a significantly lower affinity for the LM2/CDllb/CD18 complex than NIF-lFL
(Figure 18).
- - Recombinant AcaNIF9 was partially purified by reverse-phase chromatography (see Example 23). Edman degradation revealed N-E-H-D-P as N-terminal amino acid sequence confirming that correctly ~L o~,e~s ~ AcaNIF9 protein was produced. This partially purified protein was found to have a considerably higher mobility on SDS-PAGE (4-20% gradient gel; Novex) than the Pichia-produced NIF-lFL protein (30-35 kDa compared to 40-80 kDa) consistent with the presence of seven N-glycosylation sites in NIF-lFL and of only two potential N-glycosylation sites in AcaNIF9. The sample containing AcaNIF9 was tested in the competitive binding assay described in Example 1. The results demonstrated that binding of biotinylated recombinant NIF-lFL to the LM2/CDllb/CD18 complex can be prevented by recombinant AcaNIF9.

Exam~le 22 Isolation and Characterization of a NIF Protein from Ancylostoma cevlanicum.
(A) Cloninq and Seauencing of NIF Sequences From A.
cevlanicum.

WOg4/14g73 PCT~S93/12626 A full-length A. ceylanicum NIF gene was isolated by screening a cDNA library using as hybridization probe a PCR fragment effected from the same species. The PCR
fragment was obtAi~e~ using primers that target sequences which are highly conserved among the seven A.
caninum NIF isoforms described in Example lO (lFL, 3P, 2FL, 3FL, 4FL, 6FL and lP). These primers, designated YG3 and YG4, are described in Example 20.
Poly(A+) RNA was prepared from A. ceYlanicum adult worms using the QuickPrep mRNA Purification Kit (Pharmacia, Uppsala, Sweden). Using this poly(A+) RNA
preparation as template, an amplification product of about the expected length (i.e., about the same length as the PCR fragment seen with A. caninum RNA as template) was obt~in~ with the YG3/YG4 primer couple.
First strand cDNA synthesis (First-Strand cDNA Synthesis Kit; Pharmacia) and the subsequent amplification by PCR
were carried out according to the manufacturer's specifications using lO pmoles of the YG2 primer (see Example 20) and 100 ng of A. ceylanicum mRNA. The PCR
was carried out with Taq DNA polymerase (Boehringer, Mannheim, Germany), 100 pmoles of both YG3 and YG4 and using 30 temperature cycles (1 minute denaturation step at 95 C; 1 minute An~e~ling period at 55 C; 1.5 minutes elongation step). The PCR product was gel-purified and subsequently radiolabeled by "random primer extension"
(nQuickPrime Kit~; Pharmacia) for use as hybridization probe.
An A. ceylanicum cDNA library was constructed in lambda gtll using the procedures described in Example 20. The quality of the cDNA library was demonstrated by PCR analysis (Taq polymerase from Boehringer; 30 temperature cycles: l minute at 95 C; 1 ~inute at 50 C;
3 minutes at 72 C) of a number of randomly picked clones using the lambda gtll primer #1218 (New England Biolabs) in combination with an oligo(dT)-NotI primer adaptor WO94114973 PCT~S93/12626 123 21 52Sgg (Promega). The majority of the clones were found to contain cDNA inserts of variable size.
About 5xlO5 lambda cDNA clones were screened with ; the radiolabeled YG3/YG4 PCR fragment using the hybridization conditions described in Example 20.
Approximately 60 positives were identified. The cDNA
insert of one positive clone, shown by PCR analysis (see above) to contain an insert of sufficient size to encompass the entire NIF co~ing region (-850 bp), was transferred to pGEM-9Zf(-) (Promega) as a SfiI-NotI
fragment and its sequence determined. The sequence of the cDNA clone, designated AceNIF3, is shown in Figure 19 .

(B) Expression of a NIF like Protein from A. ceYlanicum in a Phage-Attached Form.
The A. ceylanicum AceNIF3 region coAing for the mature protein was cloned onto a phage display vector according to the ~o~e~ e_ described for NIF-lFL in Example 22.
The N-terminal amino acid sequence of the authentic A. ceYlanicum NIF protein is not known; it is, therefore, difficult to unambiguously locate the secretion signal ~lo~e--sing site on the deduced amino acid sequence (Figure l9). The following oligonucleotide primers were chosen to PCR amplify the AceNIF3 coding region:

YGl6:
5'-GTCGCAACTG-CGGCCCAGCC-GGCCATGGCC-GCTGACGAAC-CAACGTGCA
A-GCAG (54-mer; 5'-primer; the NcoI site and AceNIF3 N-terminus are underlined); and YGl5:
5'-GAGTTCTCGA-CTTGCGGCCG-CACCTCCGAT-AGGTGGATAA-CGGAGTGA

W094/14973 PCT~S93/12626 Z 1$2~9~ `

(48-mer; 3'-primer; the NotI site and AceNIF3 C-terminus are underlined).
Following~NcoI/~I digestion, the PCR product was gel-purified and cloned between the NcoI and NotI sites of the recipient vector. The resultant vector, designated pAN-AceNIF3, contains the intended in-frame fusion of the pelB, AceNIF3, and M13 gIII co~;ng regions.
Phages displaying the AceNIF3 protein were obtained by infecting TGl(su+) bacteria harboring pAN-AceNIF3 with M13-VCS~ 'helper'-phage (~ç~ procedures described in Example 22). The r~C~ A phages, resuspended in PBS, were assayed by an ELISA on immobilized LM2/CDllb/CD18 complex (see Figure 20). Phages displaying the A.
caninum NIF-lFL isoform (Example 22) were used as positive cGIlL~ol. Following a 30 minute inCl~h~tion with varying amounts of Pichia produced recombinant NIF-lFL, 101 virions displaying either recombinant NIF-lFL or recombinant AceNIF3 were added to the LM2/CDllb/CD18 coated wells. After a 90 minute incubation period, the amount of bound phages was detected with rabbit anti-phage serum and goat anti-rabbit alka-line phosphatase conjugate. Bi~ing of phages to the immobilized receptor (see Figure 20~ clearly indicates that the AceNIF3 protein must be displayed in a functionally active form on the phage surface. The data given in Example 22, show that phage binding occurs only when they display the NIF protein, i.e. non-displaying control phages do not bind to the LM2 monoclonal antibody nor do they bind to the LM2/CDllb/CD18 complex.
Displacement of both NIF-lFL- and AceNIF3-displaying phages by an increasing amount of soluble Pichia produced recombinant NIF-lFL demonstrates that both NIF
proteins bind to the same site on the CDllb/CD18 receptor with a comparable affinity. Phage display of the AceNIF3 protein was also demonstrated by Western WO94/14973 PCT~S93112626 blot. After fractionation on an SDS-l0~ polyacrylamide gel, phage proteins were transferred onto ProBlott membrane (Applied Biosystems Inc.) and incubated consecutively with a rabbit anti-pgIII serum (GATC GmbH, Konstanz, Germany) and goat anti-rabbit alkaline phosphatase conjugate. Bands corresponding to the wild type phage pgIII protein and to the NIF-pgIII fusion product could be visualized.

(C) Construction of ~YAM7SP-AceNIF3 and Ex~ression in Pichia.
The segment of DNA ~nco~ AceNIF3 was PCR
amplified from a subclone of AceNIF3 in pGEM-9Zf(-) (see above) using unique primers for the 5'- and 3'-ends of the coding region. The 5'-end of the proteolytically processed AceNIF3 being not unambiguously defined, a hybrid 5'-end was created based on sequence homology between AcaNIF9 and AceNIF3: the three N-terminal codons of proteolytically maturated AcaNIF9 were used as 5'-end, followed by six codons originating from the AceNIF3 sequence. The resulting N-terminal amino acid hybrid sequence was: N-E-H-E-P-T-C-K-Q, while the natural AcaNIF9 sequence was N-E-H-D-P-T-C-P-Q, and the natural AceNIF3 sequence was K-G-D-E-P-T-C-K-Q. The sequence of the 5'-primer used was 5'-AAC-GAA-CAC-GAA-CCA-ACG-TGC-AAG CAG. The 3'-primer was composed of 8 codons at the 3'-end of the coding region, a TAA stop replacing the TGA stop of the natural gene, and three unique restriction endonuclease sites (S~eI, HindIII, and XbaI). The sequence of the 3'-primer used was 5'-CCT-CCT-CCT-TCT-AGA-AGC-TTA-CTA-GTT-TAG-ATA-GGT-GGA-T
AA-CGG-AGT-GAC-G.
Amplification was accomplished using l00 pmoles of each primer, 2 units of Vent polymerase in lx Vent buffer (New England Biolabs, Beverly, MA), and 0.2 mM of each of dATP, dCTP, dGTP, and dTTP. One hundred WO94/14973 PCT~S93/12626 21S25 9g 126 nanograms of pGEM-9Zf(-) containing AceNIF3 were used as template DNA. The PCR conditions were the same for all twenty cycles: denaturation at 95 C for 1 minute, primer annealing at 60 C for 1 minute, and amplification for 1.5 minutes at 72 C. The amplification product was gel-purified and digested with SpeI.
The amplification product was ligated into StuI-SDeI cleaved pHIL7SP8 using st~n~Ard methods. The ligation mixture was used to transform E. coli WK6 selecting for ampicillin resistant clones. Based on restriction ~nd DNA sequence analysis, a correct insert sequence in one of the resulting plasmid clones, pYAM7SP-AceNI3, was selected to transform the P.
pastoris yeast strain GTS115(his4), as described in Example 12(B). Selection of His+ transformants and subsequent selection for AceNIF3 expression were performed as described in Example 12(B). The presence of AceNIF3 in Pichia cell supernatant was detected and quantified in a competitive binding assay with biotinylated NIF1 (Example 1).
Following methanol induction for 48 hours, Pichia cell supernatant was obtained by centrifugation. The crude supernatant was shown to inhibit the adhesion of human neutrophils to plastic.

Example 23 Production by E. coli of Functionally Active NIF as Either a Bacterio~ha~e-Attached Form or as 'Free' Soluble Protein (A) Cloninq of NIF-lFL on a ~hage dis~lay vector.
A phagemid-vector was assembled in which the NIF-lFL region coding for the mature protein is fused at its N-terminus to the secretion signal ~équence derived from the pelB gene and at its C-terminal end to the filamentous phage M13 gene III (gIII). This gene fuslon was placed under the transcriptional control of the Plac W094/14973 PCT~S93/12626 promoter. ~ome of the pelB codons were replaced by synonymous triplets so that the secretion signal contains an NcoI restriction site. An extra Ala-codon ; was introduced between the pelB and NIF-lFL regions such that the junction matches more closely the prokaryotic prototype signal sequence processing site. The NIF-lFL
and pgIII (product of gIII) enroAing regions are separated by (i) a linker sequence in which a NotI site is embedded and (ii) a TAG (amber) triplet which serves as a translational stop codon in a su~ strain but is frequently read as a sense codon in su+ bacterial cells.
A schematic ~e~le_cntation of the phagemid vector, designated pAN-NIF-lFL, is shown in Figure 21.
pAN-NIF-lFL was constructed by (i) PCR-amplification of the NIF-lFL co~i ng region with primers that contain 5'-extensions whose sequence is such that (ii) the NcoI/NotI directional cloning of the gel-purified PCR
fragment in the recipient vector results in the intended in frame fusion of the pelB, NIF-lFL, and gIII coding elements.
The NIF-lFL coding region was PCR-amplified making use of the following two oligonucleotide primers:

LJ045:
5~-GTCGCAACTG-CGGCCCAGCC-GGCCATGGCC-GCTAATGAAC-ACAACCTGA
G-GTGC (54-mer; 5'-primer; The NcoI site and NIF-lFL
N-terminus are underlined) LJ046:
5~-GA~l-l~.CGA-CTTGCGGCCG-CAG~lAA-CTCTCGGAAT-CGATAAAAC
T-C (51-mer; 3'-primer; the NotI site and NIF-lFL
C-terminus are underlined) ~-The NIF-lFL region and flAnking seqffences present in pAN-NIF-lFL were entirely sequenced to rule out the presence of unwanted mutations.

W094114973 PCT~S93/12626 2 ~.S?,599 ` ~ - 128 (B) Dis~laY of functional NIF bY filamentous ~haqes.
In su+ bacteria such as TGl, the pAN-NIF-lFL
phagemid-vector has thë potential to code for a NIF-lFL-pgIII fusion protein. When the TGl[pAN-NIF-lFL]
host cells are infected with a so-called 'helper'-phage, this fusion protein can, during morphogenesis, become incorporated into filamentous virions (both 'helper'-phages and pC~ o-virions which encapsidate one specific strand of the phagemid). Phage particles were rescued with Ml3-VCS 'helper'-phage (Stratagene) infection as follows. A l ml culture of TGltpAN-NIF-lFL] grown at 37 C in 2xTY (2xTY: Tryptone 16 g/L; Yeast extract lO g/L; NaCl 5 g/L) cont~;n;ng lOO
~g/ml carbenicillin (or ampicillin) and 1% glucose to a density of OD~m ~ 0.5-0.6 is infected with Ml3-VCS at a multiplicity of infection of -20. The infected culture is incubated at 37 C for 30 minutes without shaking and then for another 30 minutes with ~hAki~g. A lO ml prewarmed 2xTY aliquot containing both carbenicillin (lOO ~g/ml) and kanamycin (50 ~g/ml) is inoculated with the l ml infected culture. The mixture is incubated with shaking first for 60 minutes at 37-C and then overnight at -30 C. After removal of the infected cells by centrifugation l:5 volume 20% polyethyleen glycol/2.5 M NaCl is added to the supernatant. Following a 60 minute incubation on ice, the precipitated phages are collected by centrifugation and resuspended in PBS
(Na2HPO4.2H20 l.14 g/L; KH2PO4 0.2 g/L; NaCl 8.Og/L; KCl 0.2 g/L; pH 7.3).
The rescued phages were shown to display functionally active NIF in several assays: (A) Western blot (After fractionation by SDS-10% PAGE, phage proteins were transferred onto ProBlott (Applied Biosystems Inc., Foster City, CA) membrane and incubated with rabbit anti-phage serum and goat anti-rabbit alkaline phosphatase conjugate. A band corresponding to W094/14973 PCT~S93/12626 129 21~2 59 9 the NIF-pgIII product could be visualized); (B) CDllb/CD18-ELISA (see Figure 22) (NIF-phage were then assayed for binding to CDllb/CD18 (non-displaying phage were used as negative control). CDllb/CD18-coated wells were prepared either by direct immobilization using 0.25 ~g/ml immunopurified CDllb/CD18 receptor (Diamond et al., 1990, J. Cell Biol., 111, 3129-3139), or by immuno-capture with monoclonal antibody LM2 (ATCC
number: HB 204). Binding of phages was detected with rabbit anti-phage antiserum and goat anti-rabbit alkaline phcsphatase conjugate. Binding of phages to the immobilized t e~ or was shown to occur only when they display the NIF protein. It was also shown that NIF-phage are not able to bind to the LM2 monoclonal antibody nor to the CDllb/CD18-coated wells after a pre-incubation with lmM Pichia-produced recombinant NIF-lFL for 30 minutes); (C) 3D2-ELISA (3D2 is a non-neutralizing mouse monoclonal antibody specific for NIF (see Example 26). In ~GI,L~ast to non-displaying control phages, NIF-phage were found to bind to 3D2-coated wells. NIF-phage binding could be eliminated by either blocking the 3D2-wells with 1 mM
Pichia-produced recombinant NIF-lFL or blocking the NIF-phages with 1 mM 3D2 monoclonal antibody); and (D) Panning aqainst CDllb/CD18 (pAN-NIF-lFL phage (101 virions) were mixed with an equal amount of irrelevant non-displaying phage (fd-tet; 10~virions), diluted in 100 ~1 Binding Buffer (PBS, 1 mM CaCl2, 1 mM MgCl2, 0.4%
Tween-20 and 2% Skim-Milk) and incubated in a CDllb/CD18-coated microtiter-well. After incubation for 120 minutes, and wAchi~g with PBS cont~;ning 1 mM CaCl2 1 mM MgCl2 and 0.4% Tween-20 ten times, bound phage were eluted during a 10 minute incubation wi~ glycine-HCl pH
2Ø Following neutralization with 1 M Tris-HCL pH 8.0, the number of tetracycline resistant (fd-tet) and ampicillin resistant (pAN-NIF-lFL) colony forming units WOg4/14973 PCT~S93/12626 ~,~S?.599 was determined; pAN-NIF phage were 30-fold enriched over the non-displaying fd-tet phage).
The above experiments, e.g. functional display of NIF-lFL on filamentous phage and the specific enrichment of such NIF-phages by binding selection, show it is possible to use the phage technology for the identification of higher-affinity NIF variants (i.e., both naturally occuring isoforms or engineered mutants).
Similar to what has been done in the immunoglobulin field, it should be possible to clone the vast majority of the A. c~ninum NIF protein repertoire on phage and then to select the highest affinity NIF isoform by subjecting the phage library to several consecutive b; nA ing selection cycles (pAnni ng) using the CDllb/CD18 receptor as target. Our sequence data show that the extent of conservation of the 5' and 3' termini of the region enco~i~g mature NIF allows the design of (degenerate) oligonucleotide primers to rescue a substantial part of the NIF protein repertoire. For example, we generated PCR-amplification fragments of the expected length using a lambda DNA preparation of the pooled A. caninum cDNA library as target with the following oligonucleotide primer sets:
5'-~rimer targetina the N-terminus: an eauimolar mixture of three primers that contain at their 3'-end the following matching seauences:
AAT-GAA-CAC-AAC-CTG-ASG-TGC-3' AAT-GAA-CAC-GAC-CCA-ACG-TGT-3' AAT-GAA-CAC-AAA-CCG-ATR-TGC-3' where S = C or G and R = A or G; and 3'-primer tarqetina the C-terminus: an eauimolar mixture of two primers that contain at their 3'-end the following matc~ing se~nceC
TAA-CTC-TCG-GAA-TCG-ATA-AAA-3' TAA-CTC-TCG-AAA-CSG-ATA-AAA-3' where S = C or G.

W094/14973 PCT~S93112626 2lls2599 The PCR primers contain 5'-extensions which incorporate restriction sites allowing the facile unidirectional cloning of the amplification product in - an appropriate display vector.

(C) Secretion of Soluble and FunctionallY Active NIF.
The phagemid display vector cont~;ni~g the NIF
gene, pAN-NIF-lFL, is suitable for the production of NIF-lFL in both a phage-attached form and as 'free' soluble protein. In su bacteria such as WK6, the pAN-NIF-lFL phagemid-vector has the potential to direct the synthesis of the NIF-lFL protein in a 'free' (i.e., not phage-attached) form.
Overnight induction of the Plac promoter by addition of iso~G~yl-~-D-thiogalactopyranoside (1 mM
final concentration) to a WX6[pAN-NIF-lFL] culture was found to result in the accumulation of CDllb/CD18-binA;~g activity as shown by ELISA (on LM2/CDllb/CD18 plates; detection was done with HRP-conjugated monoclonal antibody 3D2). The recombinant NIF-lFL protein could be detected in both the supernatant of the induced culture and in a total cell lysate prepared by sonication of the induced cells followed by a clearing step. Comparison of the ELISA-signal with that generated by known amounts of Pichia-produced rNIFl allowed us to estimate that the rNIF protein accumulates to about lmg per liter of E.
coli culture. The rNIF protein was also immunopurified on a 3D2-Emphaze column (see Example 27) starting from a French-Press lysate of induced WK6[pAN-NIF-lFL] cells.
Material eluted at low pH was still active as determined by the LM2/CDllb/CD18 ELISA. The E. coli rNIF protein was shown to migrate as a sharp band on SDS-polyacrylamide gel and could be detected by rabbit anti-Pichia-rNIFl serum in immuno-blot analysis.

WO94114973 PCT~S93112626 2~S~59~ 132 Exam~le 24 Alternative Purification Method For Pichia Produced NIF.
Cell-free supernatant was filtered (0.2 ~m) and submitted to a diafiltration on a polyethersulfone omega membrane (30kDa cut-off; 0.75 ft2; Filtron) with lO
volumes of 50 mM citric acid pH 3.5 contA;n;ng lmM EDTA.
After adjustment to pH 7.4 by A~; ng l M Tris-HCl, the solution was left on ice for at least one hour.
Precipitated material was removed by filtration (0.2 ~m). The cleared supernatant was submitted to a second dialfiltration (lO volumes lO mM phosphate pH 7.4).
Afterwards calcium chloride was added to a final concentration of 0.3 mM. The solution was applied on a MacroPrep (40 ~m) Hydroxyapatite (Bio-Rad Laboratories) column equilibrated with lO mM phosphate pH 7.4 and containing 0.3 mM CaCl2. After washing with 5 column volumes of the equilibration buffer, the recombinant NIF
protein was eluted with 90 mM phosphate pH 7.4.
Fractions contA;n;~g recombinant NIF were identified by binding assays on LM2/CDllb/CDl8 plates and pooled.
Subsequently, the protein present in the pooled fractions was further purified by reversed phase chromatography on a Poros Rl/H (Perseptive Biosystems) column equilibrated with lO mM ammonium formate pH 6.4 and 10% acetonitrile. Recombinant NIF was eluted by increasing the acetonitrile concentration. The fractions containing NIF were identified by gel-electrophoresis and were pooled. Acetonitrile present in this pool was removed in a rotavapor before freeze-drying. The dry protein was redissolved in PBS and applied on a Superdex 200 (Pharmacia) gel filtration column equilibrated in PBS. Fractions contA;n;~g the NIF protein were pooled and concentrated by ultrafiltration on an omega membrane (lO kDa cut-off; Filtron). The recombinant NIF protein was stored at -80 C.

WO94/14973 PCT~S93/12626 -Example 25 Expression of Functional Derivatives of NIF-lFL in Pichia ~astoris.
(A) pMa5-hNIFl and pMcS-hNIF1 Expression Constructs.
The segment of DNA encoding NIF was PCR amplified from a subclone of NIF-lFL in BluescriptII (Stratagene, La Jolla, CA) using unique primers for the 5'- and 3'-ends of the coding region.
The 5'-primer was composed of two restriction sites (EcoRI and HpaI) and the 23 first nucleotides of the region begi~ning at the 5'-end of proteolytically processed NIF and the sucre~in~ 8 codons. The codon for the first residue of the mature NIF was altered from AAT to AAC (both codons translate to asparagine) and constitutes part of the HpaI restriction sites (GTT/AAC). The sequence of the 5'-primer used was 5'-CCG-GAA-TTC-GTT-AAC-GAA-CAC-AAC-CTG-AGG-TGC-CC. The 3'-primer has been described in Example 12(B).
Amplification was accomplished using 100 pmol of each primer, 2 units of Vent polymerase in lX Vent buffer (New England Biolabs, Beverly, MA), and 0.2 mM of each of dATP, dCTP, dGTP, and dTTP. One hundred nanograms of BluescriptII-cont~inin~ NIF-lFL were used as template DNA. The PCR conditions were the same for all twenty cycles: denaturation at 95 C for 1 minute, primer annealing at 60-C for 1 minute, and amplification for 1.5 minutes at 72 C. The amplification product was gel-purified and digested with EcoRI and HindIII.
The amplification product was then ligated into EcoRI-HindIII cleaved pMa5-8 and pMc5-8 respectively [Stanssens et al., Nucl. Acids Res. 17: 4441-4454 (1989)], using standard methods. The ligation mixtures were used to transform competent E. coli WK6 (Zell et al., (1987) EMBO J., 6: 1809-1815). Cells resistant to ampicillin and chloramphenicol, respectively, were selected and obtained on appropriate plates. Based on W094/14973 PCT~S93/12626 restriction and DNA sequence analysis, a correct insert sequence in each of the resulting plasmid clones, pMa5-hNIF1 and pMc5-hNIFl, were selected.

(B) Construction of pMa5-hNIFl/~Gl1-5.
The NIF-lFL protein contains seven potential N
(Asparagine)-glycosylation sites (consensus N-X-T/S
amino acid sequence).
pMa5-hNIFl/~Gl1-5 is a derivative of pMa5-hNIFl (see above) in which five potential N-glycosylation sites of NIP-lFL have been modified by substituting glutamine residues for each of the asparagine residues in the corresponding consensus sequences. These residues are Asnl, Asn~8, Asn87, Asnl~, and Asn~30, where the number in superscript corresponds to the amino acid residue number of NIF-lFL (see Fig. 8).
Stepwise site-directed mutagenesis was performed following the methodology described in Stanssens et al., (1989), Nucl. Acids Res. 17: 4441-4454, and using the following oligonucleotides:
(I) ~Gll:dCCGGGCATTTCGGTACCTTGCTGCGGGCACCTC, (II) ~Gl2:dCCTAATCGA~~ G~ACCCGGGCA~ ~llCC, (III)~Gl3:dAA~ CGAGCA~ ~lGCACTCATGTAGGCG~ C, (IV) ~Gl4:dCAGAGCTTr~r.Ar.A.~G~ll~GA~L~ G, and (V) ~Gl5:dCT~l.~ll-L~ GCAGGTTGAAAGCCTC.
In the first mutagenesis round, the oligonucleotides I, IV and V were annealed together to the single strand DNA (ssDNA) template pMaS-hNIFl to modify the corresponding glycosylation sites Gll, Gl4 and Gl5. A resulting plasmid clone having the three intended sites altered was then used to prepare ssDNA
template for the next mutagenesis round in order to modify the Gl2 and Gl3 remaining sites ~sing the appropriate oligonucleotides (II and III).

(C) Construction of pMa5-NIF-lFL/~h,Gl6-7.

W094/14973 PCT~S93/12626 -- 21 5259g The strategy outlined in (B) above was performed in parallel to construct another NIF-lFL derivative, pMa5-NIF-lFL/AhG16-7, in which the potential N-glycosylation sites G16 and G17 of NIF-lFL have been modified by substituting glutamine residues for each of the asparagine residues in the corresponding consensus sequences. These residues are Asnl~, and Asn~. In addition, the HpaI restriction site (GTTAAC) present in the NIF-lFL coding sequence was removed by introducing a silent mutation at the appropriate position: the AAC
codon for As~ was replaced by a AAT codon.
Stepwise site-directed mutagenesis was performed following the methodology described in Stanssens et al., (1989), Nucl. Acids Res. 17: 4441-4454, and using the following oligonucleotides:
(VI) ~G16:dCGG~ CA~~ l~lA ~ GGGTAGTGGC, (VII) ~G17:dGGATCCG~GA~,lC~ GTCTG~ , and (VIII) ~h :d~L~C~A~G~GCAAT~A~CTGCGC.
In the first mutagenesis round, the oligo-nucleotides VII and VIII were annealed together to thessDNA template to modify the glycosylation site Gl7 and to remove the H~aI restriction site. A resulting plasmid clone harbouring the two intended sites altered was then used to prepare ssDNA template for the next mutagenesis round in order to modify the Gl6 remaining site using the appropriate oligonucleotide tVI).

(D) Construction of ~MaS-hNIF1/Ah.Gl1-7.
The NIF-lFL derivative hNIFl/~h,Gl1-7 was constructed using stAn~Ard methods by combining appropriate fragments prepared from the vectors pMa5-hNIF1/AG11-5 (prepared as in (B) above) and pMa5-NIF-lFL/Ah,G16-7 (prepared as in (~ above). The 361 bp AaeI-HindIII fragment prepared from the vector pMa5-NIF-lFL/~h,G16-7, and containing the three substitutions described in (C) above, was cloned into W094/14973 PCT~S93/12626 -the large AaeI-HindIII vector fragment prepared from the vector pMaS-hNIF1/~G11-5, replacing the corresponding 361 bp AgeI-HindIII wild type NIF-lFL fragment of this vector.
The preC~nce of the seven Asn/Gln substitutions as well as of the modified H~aI restriction site (Asn~
codon modification) in the resulting plasmid, pMa5-hNIFl/ h,G11-7, was confirmed by sequencing analysis of the complete NIF insert. A one base pair deletion in the NIF sequence (a missing G nucleotide in the Gly20l GGA codon) revealed by this sequence analysis was corrected by site directed mutagenesis using the oligonucleotide dGTAAATCGG~ llCA~ ~ lG.

(E) Construction of DYAM7SP-hNIFl/~G11-5 and Expression in Pichia ~astoris.
The segment of DNA ~ncoAi~ hNIFl/~G11-5 was PCR-amplified from a subclone of pMa5-hNIFl/~G11-5 following the methodology described in Example 12(B) and using the same set of primers. After purification, the amplification product was digested with SpeI and ligated into StuI-SpeI cleaved pHIL7SP8 using st~nA~d methods.
The ligation mixture was used to transform E. coli WK6, and ampicillin resistant clones were obtained on ampicillin plates. Based on restriction and DNA
sequence analysis, a correct insert sequence in one of the resulting plasmid clones, pYAM7SP-hNIFl/~Gll-5, was selected to transform the P. ~astoris yeast strain GTS115(his4), as described in Example 12(B). Selection of His+ transformants and subsequent selection for NIF-lFL/~G11-5 expression were performed as described in Example 12(B). The presence of NIF-lFL/~G11-5 in Pichia cell supernatant was detected and quant~fied using the LM2/CDllb/CD18 based ELISA with 3D2-HRP detection (see Example 1).

WOg4/14973 PCT~S93/12626 215259g (F) Construction of ~YAM7SP-hNIFl/~h G11-7 and ex~ression in Pichia pastoris.
The HpaI-SpeI fragment of DNA encoding hNIF1/ah,G11-7 was prepared from the vector pMa5-hNIFl/ah,G11-7 and ligated into StuI-SpeI cleaved pHIL7SP8 using st~n~rd methods. The ligation mixture was used to transform E. coli WK6, and ampicillin resistant clones were obtAin~ on ampicillin plates.
Based on restriction and DNA sequence analysis, a correct insert sequence in one of the resulting plasmid clones, pYA~7SP-hNIFl/~h,G11-7, was selected to transform the P. pastoris yeast strain GTS115(his4), as described in Example 12(B). Selection of His+
transformants and subsequent selection for NIF-lFL/~h,G11-7 expression were performed as described in Example 12(B). The prPsenc~ of NIF-lFL/~h,G11-7 in Pichia cell supernatant was detected and quantified using the LM2/CDllb/CD18 based ELISA with 3D2-HRP
detection (see Example 1).

(G) Purification and Characterization of Recombinant NIF-lFL/~G11-5 and Recombinant NIF-lFL/~h G11-7.
Following methanol induction for 48 hours, Pichia cell supernatants were obtained by centrifugation for 15 minutes at 1,800 x g.
Recombinant NIF-lFL/~G11-5 was purified exactly as described in Example 23. The recombinant NIF-lFL mutant was found to migrate with an apparent molecular weight of 36-50kDa on SDS-PAGE (4-20% gradient gel; Novex) under non-reducing conditions. The band is more discrete and has a significantly higher mobility than wild type NIF-lFL produced in Pichia. Recombinant NIF-lFL/~h,G11-7 was purified by hydrox~apatite and reverse-phase chromatography (see Example 23; the gelfiltration step on Superdex was omitted). Under non-reducing conditions, the purified protein was found W094/14973 PCT~S93/12626 -to migrate as a single band on SDS-PAGE (4-20% gradient gel; Novex), with an apparent molecular weight of about 3OkDa. The observed higher mobility and apparent lesser heterogeneity of NIF-lFL/~G11-5 and NIF-lFL/~h,Gl1-7 compared to NIF-lFL is likely due to the relatively decreased extent of glycosylation of these mutants compared to the wild-type proein.
Both mutants were evaluated in the plastic adhesion assay (see Figure 23). The results indicate that elimination of part or all of the seven potential N-glycosylat-ion sites does not affect the potency of the NIF-lFL molecule as measured in this in vitro assay.

Exam~le 26 Pre~aration of Monoclonal Antibodies to NIF
Hybridomas producing MAbs that bind to NIF were prepared from mice immunized with Pichia ~LGduced recombinant NIF-lFL by previously described methods (H.R. Soule, E. T~ ~r, T.S. Edgington, Proc. Natl.
Acad. Sci. USA 80:1332 (1983); G. Kohler and C.
Milstein, Nature (London) 256:495 (1975)).
Female balb-c mice between the ages of 8 to 15 weeks were inoculated subcutaneously with 10 ~g of recombinant NIF (from Example 12) in Complete Freund's Adjuvant, then 3 weeks later were inoculated subcutane-ously with 10 ~g of the recombinant NIF in IncompleteFreund's Adjuvant, then 2 weeks later were inoculated interperitoneally with 10 ~g of the recombinant NIF in 4 mg of alum, then three to four weeks later and also 4 days prior fusion were interperitoneally innoculated with lo ~g of the recombinant NIF in saline. Mice - producing polyclonal antibody to recombinant NIF were determined by immunoassay of their seru~ using l25I
labeled recombinant NIF and immobilized goat anti-mouse IgG. Mice having a titer between 1:25,000 to 1:50,000 were selected. Spleen cells were isolated from the mice WO94114973 PCT~S93/12626 -- ~1 52599 and were fused with tumor cells in a 5:1 ratio of spleen cells to tumor cells. Hybridoma cells expressing monoclonal antibody b;n~i~g to NIF were selected by the same immunoassy. Monoclonal antibody was expressed from the selected hybridoma cells by inoc~ tion interperi-toneally of 5 x 106 hybridoma cells into pristane-primed female balb-c mice.
One monoclonal antibody designated 3D2 was shown to bind both hookworm-derived NIF and Pichia-produced recombinant NIF-lFL by antigen capture assay (E. Harlow and D.P. Lane, Antiho~ies: A Laboratory MA~ 7 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988), pp. 192-193).

Example 27 CouDlina of 3D2 Murine Monoclonal Antibodv to 3M Em~haze Biosu~ort Medium (A) Sinqle SteD Method.
Twenty eight milligrams of 3D2 antibody was concentrated to 0.5 ml and 4.5 ml of 0.6 M sodium carbonate, 0.1 M sodium citrate, pH 9.0 was added.
Three hundred fifteen milligrams of dry Emphaze Biosupport Medium (3M, St. Paul, Minnesota) was added to the antibody solution, and mixed end-over-end for one hour. The Emphaze slurry was collected over a plastic frit, draining the remaining antibody solution. The resin was washed with 20 ml of 10mM sodium phosphate, 0.15 M sodium chloride, pH 7.3. The collected resin was then mixed end-over-end with 3.0 M ethanolamine, pH 9.0 for 2.5 hours. The resin was again collected on a frit, and washed with 10 mM sodium phosphate, 0.15 M sodium chloride, pH 7.3 until the solution flowing through the frit was pH 7.3. The resin was stored-~n 10mM sodium phosphate, 0.15 M sodium chloride, 0.05% sodium azide, pH 7.3, until use. Two millilters of 3D2/Emphaze resin WO94/14973 PCT~S93112626 -~.52599 140 coupled using this procedure was found to bind 1.4 mg of purified NIF protein.

(B) Two Step Method.
Thirty milligrams of 3D2 antibody was dialyzed into 0.5 M Tris-HCl, pH 4.0, concentrated to a volume of 5.0 ml (6 mg/ml), and chilled to 4-C. Three hundred fifteen milligrams of Emphaze Bio_u~o~- Medium was added to the antibody solution and mixed end-over-end for 10 minutes at 4 C. Sodium sulfate was added to a concentration of 0.8 M (563 ~g) and the Emphaze slurry was mixed end-over-end for an additional 10 minutes at 4 C. The pH was raised to 9.0 by dropwise addition of lM NaOH.
The coupling reaction was allowed to proceed for 60 minutes at 4-C with end-over-end mixing. The Emphaze slurry was collected over a plastic frit, and remaining antibody solution was drained. The resin was washed with 20 ml of 10mM sodium phosphate, 0.15 M sodium chloride, pH 7.3. The reaction was ~Pnch~A by the addition of 6 ml of 1.0 M ethanolamine, pH 9.3, and the resin in ethanolamine was mixed end-over-end for 2.5 hours at 4 C. The resin was again collected over a frit, and washed with 0.2 M sodium phosphate, 0.5 M
sodium chloride, pH 7.3 until the solution flowing through the frit was pH 7.3. The resin was suspended in 0.01 M sodium phosphate, 0.15 M sodium chloride, 0.05%
(w/v) sodium azide, pH 7.3, until use. A 2ml 3D2/Emphaze column coupled using this method typically bound 1.5 mg of purified NIF protein.

(C) Scale-U of Coulinq Reactions.
Twenty milliliters of resin has been made using both coupling methods by simply scaling~up all volumes and amounts 10 fold. 8Oth methods yielded resin capable of binding NIF protein. A 20 ml portion of 3D2/Emphaze resin coupled by the Two Step Method bound 10 fold more WO94/14973 PCT~S93112626 -141 ~1 5~599 NIF protein than did 2 ml columns (15-19 mg versus 1.5 mg), whereas 20 ml of 3D2/Emphaze resin coupled using the Single Step Method did not scale linearly (typically 5-8 mg NIF protein bound). Thus, the Two Step Coupling Method was used for larger couplings.
A 150 ml Two Step coupling was performed in which 2.25 g of 3D2 antibody in 450 ml of 0.5 M Tris-HCl, pH
4.0 at 4 C was mixed with 19 g of Emphaze Bio~ ol~
Medium in a 1000 ml microcarrier spinner flask (Bellco Glass Inc., Vineland, New Jersey) for 10 minutes at 4 C.
Forty two g~ams of sodium sulfate was added to the Emphaze slurry and this was stirred for an additional 10 minutes at 4 C. The pH was raised to 9.0 by dropwise addition of 1 M NaOH through the arms of the spinner flask. The reaction was allowed to proceed for 60 minutes at 4 C with stirring. The Emphaze slurry was collected over a 90 mm, 0.45 micron filter (Corning Glass Works, Corning, New York), and remaining antibody solution was drained. The resin was washed with 1 liter of 0.01 M sodium phosphate, 0.15 M sodium chloride, pH
7.3. The reaction was qllPnchP~ by the addition of 500 ml of 1.0 M ethanolamine, pH 9.3 to the resin. The quenching reaction was allowed to continue 2.5 hours at 4 C with stirring. The resin was again collected over a 90mm, 0.45 micron cellulose acetate filter, and washed with 0.2 M sodium phosphate, 0.5 M sodium chloride, pH
7.3 until the solution flowing through the filter was pH
7.3. The resin was susp~n~PA in 0.01 M sodium phosphate, 0.15 M sodium chloride, 0.05% sodium azide, pH 7.3, until use. A 2 ml portion of res-n from this coupling bound 1.5 mg of NIF protein, the same amount of ~ NIF protein bound by resin coupled in a 2 ml reaction.

Example 28 Purification of Recombinant NIF Protein Usinq 3D2/EmPhaze ImmunoaffinitY ChromatoqraPhY Column WO94/14973 PCT~S93/lt626 ~

; 142 Cell supernatant containing recombinant NIF protein was filtered through a Sartobran PH 0.07 micron dead-end filter (Sartorius North America, Bohemia, New York), and then concentrated 10-50 fold by tangential flow filtration using a Mini Crossflow System contA;n;ng 10 kDa Minisart polysulfone membrane modules (Sartorius).
The concentrate was then diafiltered against five volumes of 0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.3 in the Nini Crossflow apparatus.
Immediately before application to the 3D2/Emphaze column, the soncentrate was filtered through a 90mm, 0.22 micron cellulose acetate filter (Corning).
Approximately 150 mg of NIF protein was applied to a 400 ml 3D2/Emphaze column at 20 ml/min. The concentrate was washed from the column with 400 ml of 0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.3 at 20 ml/min.
The column flowthrough was collected and retained. The column was then washed with 400 ml of 1 M NaCl and the wash was discarded. The recombinant NIF protein bound to the column was eluted by applying 800 ml of 0.1 M
glycine, pH 2.5. After elution, the purified NIF
protein from the column was brought to neutral pH by the dropwise addition of 1 M Tris base. The column was then re-equilibrated to loading conditions by passing 800 ml of 0.1 M sodium phosphate, 0.15 M sodium chloride, pH
7.3, through it until the pH of solution exiting the column was pH 7.3.
When approximately 1 g of NIF protein had been purified by the 3D2/Emphaze column, the protein was pooled and then concentrated using an Easyflow 20 kDA
polysulfone concentration apparatus (Sartorius). The concentrated protein was then applied at a flow rate of 10 ml/min. to a 60 cm x 600 cm Superde~ 200 prep gel filtration column (Pharmacia, Piscataway, New Jersey) 3~ equilibrated in 0.01 M sodium phosphate, 0.15 M sodium W094/14973 PCT~S93112626 '_ 143 2I 52 5~

chloride, pH 7.3. The only peak observed during elution ; (870 - 1050 ml) corresponds to NIF protein.

Example 29 Neutrohil InhibitorY Factor is an Inhibitor of Eosinophil Adhesion to Vascular Endothelial Cells NIF was assayed for effect on adhesion of human eosinophils to cytokine-stimulated endothelial cells.
Eosinophils were isolated from normal individuals as described by Moser et al (1992a) ~J. Immunol.
lo 149:1432-143~]. Isolated eosinophils were cultured in the presence of 10 pM GM-CSF and 10 pM IL-3 for 24 hours following the procedure of Moser et al (1992a).
Endothelial cells were harvested from umbilical cord veins, seeded in tissue culture flasks and transferred to 24-well plates as described by Moser et al (1992a).
The adhesion assay was carried out following the procedure described by Moser et al (1992a). Briefly, human umbilical vein endothelial cell (HUVEC) monolayers were washed with Hank's balanced salt solution (HBSS) and preincubated with 500 ~l of TNF~ at a final concentration of 10 ng/ml for 4 hours at 37 C.
Immediately before use in adhesion assays, HUVEC
monolayers were washed. Next, 2.5 X 105 eosinophils in 500 ~l of HBSS cont~in;ng 5 mg/ml of purified human albumin were layered onto the washed HUVEC monolayers.
After incubation for 30 minutes at 37 C and saturated humidity/5% C02, the 24-well plate was submerged three times in a bath of 300 ml PBS to remove loosely adherent eosinophils. Plates were dried at 4-C and the number of adherent neutrophils was quantitated by measuring peroxidase activity, as described in Moser et al, 1992b [Blood 79:2937].
Recombinant NIF (rNIF) inhibited adhesion of GM-CSF/IL-3 primed human neutrophils to TNF-activated H W EC monolayers, to a maximum of approximately 63~

WO94/14973 PCT~S93/12626 ?,~.S~599 144 inhibition at 100 nM rNIF. About 50% inhibition of adhesion was obt~;~e~ in the presence of approximately 10 nM rNIF tsee Figure 13).

Example 30 Bindinq of NIF to Leukocytes The interaction of NIF with leukocytes was assessed by flow cytometry using biotinylated recombinant NIF.
Biotinylated rNIF was prepared as described above for derivatizati~n of NIF purified from hookworm homogenates with the exception that the rNIF was derivatized at a final biotin-LC-hydrazide concentration of 0.14 mM. A
buffy coat preparation of human leukocytes, suspended in 2% newborn calf serum, 1% NaN3 (PBS-NCS), was incubated with biotinylated rNIF (0.6 ~g/ml ) for 5 minutes at room temperature. Red blood cells were lysed by addition of 3 ml 150 mM NH4Cl, 1% NaN3 for 5 minutes at room temperature. The cells were washed twice in 1 ml PBS-NCS and resusp~n~e~ in 50 ~1 wash buffer (5% newborn calf serum, 0.1% NaN3 in PBS) cont~in;ng 0.25 ~g/ml streptavidin-phycoerythrin (Pharmingen). After 15 minutes at 4 C the cells were washed and resuspended in 0.5 ml wash buffer. Flow cytometry was performed with a FACScan instrument (Becton Dickinson) using Lysys II
software (Becton Dickinson). Leukocyte populations were electronically gated using cytograms of ~orward versus right angle light scatter. Background binding was determined using biotinylated BSA (Pierce).

W094/14973 PCT~S93/lt626 -145 21 52~99 The binding of biotinylated rNIF to lymphocytes - (panels A, D and G), monocytes (panels B, E and H) and granulocytes (panels C, F and I) was analyzed by flow cytometry. (See Figure 24). Cell types were electroni-cally gated by using cytograms of forward versus right angle light scatter. Panels A-C illustrate negative control reactions using biotinylated BSA, panels D-F
show reactivity with biotinylated rNIF and panels G-I
show reactivity with biotinylated rNIF in the presence lo of a 50-fold molar ~Y5~CC of underivatized rNIF. All reactions were developed with ~L~Lavidin-phycoerythrin. Cell number is given on the ordinate and fluorescence instensity on the abscissa.
Greater than 95% of the gated granulocytes and monocytes were found to bind biotinylated rNIF relative to the BSA-biotin control (Fig. 24). In contrast, rNIF
associated with a discrete minor population of peripheral blood lymphocytes. Cellular binding of NIF
was specific because co;nsl~h~tion of biotinylated rNIF
and a 50-fold molar ~YC c~ of non-derivatized rNIF
abolished the reaction with all three leukocyte cell populations (Fig. 24). These data demonstrate that in each of these leukocyte populations there exist cells that specifically bind NIF.

Example 31 Direct concordance of NIF bindinq and CDllb/CD18 expression WO94114973 PCT~S93/12626 ~,~S~F~99 146 Dual reaction flow cytometry experiments were done to investigate CDllb/CDl8 expression of the lymphocyte population that bound NIF, using a monoclonal antibody to CDllb/CDl8 (LM2; ATCC# HB204). Leukocyte staining with biotinylated rNIF was done as described in Example 30. In these experiments LM2 (1 ~g/ml) was incubated with the whole leukocyte preparation in addition to biotinylated rNIF. After red blood cell lysis and washing, leukocytes were reacted with FITC-conjugated goat (Fab') 2 anti-mouse IgG (Caltag) at 35 ~g/ml in addition to ~L~e~Lavidin-phycoeLyLhrin.
Direct concordance of NIF b;~;ng and CDllb/CDl8 expression was deomnstrated by dual fluorescence analysis by flow cytometry of peripheral blood lymphocyte populations (electronically gated by using cytograms of forward versus right angle light scatter) for rNIF binding versus binding of anti-CDllb/CDl8 monoclonal antibody LM2. All cells reactive with rNIF
were positive for CDllb/CDl8. (See Figure 25).
These experiments demonstrated a direct concordance between lymphocytes that bound NIF and those that expressed CDllb/CDl8 (Figure 25). These results provide strong evidence that the integrin CDllb/CDl8 is a binding site for NIF on leukocytes.

Example 32 NIF Binds the I-domain Portion of the CDllb PolY~e~tide WO94/14973 PCT~S93tl2626 147 2I 5259g The I-domain (=A domain) portion of the CDllb/CD18 integrin comprises a segment of the CDllb polypeptide which is approximately bounded by the residues Cys'~ and Glycine~' (Mic~;shita, M., Videm, V., and Arnaout, M.A.
(1993) Cell 72,857-867). To demonstrate direct interaction between NIF and the I-domain peptide, we expressed the I-domain peptide as a recombinant soluble fusion peptide in E. coli cells. The fusion is a 8 amino acid tag peptide termed FLAG The PCR primers used to clone I-domain were based on amino acid seqll~nc~s of the CDllb polypeptide (Glyl'l-Ala3's). The primer se~nceC were 5'-CCA-AAG-CTT-GGA-TCC-AAC-CTA-CGG-CAG-CAG-CC-3' (primer 1), and 5'-CCA-TCT-AGA-CGC-AAA-GAT-CTT-CTC-CCG-AAG-CT-3' (primer 2). Primer 1 contains an additional 5'-HindIII restricion endonuclease site, and primer 2 contains an additional 3'-XbaI site. The primers were used pairwise with random-primed single-stranded cDNA (cDNA Synthesis Plus, Amersham) synthesized from human monocyte total RNA (see Example 10 for protocols). The starting material was -0.5 g human monocytes. PCR conditions were:
denaturation at 95 C for 30 seconds, annealing at 45 C
for 30 seconds, elongation at 72 C for 2 minutes, for 30 cycles. All PCR reagents were from Perkin-Elmer.
PCR amplification produced a fragment containing a -615 base pair I-domain coA; ng region with appropriate 5'-and 3'-restriction sites. The HindIII/XbaI-digested WO94/14973 PCT~S93/12626 -'599 ~ ` 148 fragment was ligated into the NindIII/XbaI-cleaved plasmid vector pFLAG-l (International Biotechnologies, Inc). The construct was used to transform Epicurean Coli SURE competent cells (Stratagene), which were plated on LB media agar plates containing 100 ~g/ml ampicillin and 0.4% glucose for selection. Three milliter overnight cultures inoculated with transformed colonies were grown at 37-C in LB + 100 ~g/ml ampicillin. Isolation of plasmid DNA from the overnight cultures was performed with a Magic Miniprep kit (Promega), and isolated plasmid DNA was restricted with ~indIII and X~aI. Digestion products of -615 and -5370 bp were observed, indicating the presence of the CDllb I-domain-encoding insert in the expression vector. This expression plasmid was termed PMl. Five hundred milliters of LB + 100 ~g/ml ampicillin was inoculated with 5 ml of an overnight culture of E. coli transformed with the plasmid PMl. The 500 ml culture was grown at 37 C to an OD~ of 0.400 (-120 minutes), induced with 1.7 mM iso~-Gpyl b-D-thiogalactopyranoside (Sigma), and grown for two additional hours at 37 C. Cells were harvested by centrifugation at 5,000 g for 5 minutes, 25 C. The cells were then resuspended in 50 ml of an extraction buffer-l (50 mM Tris, pH 8.0, 5 mM EDTA, 0.25 mg/ml lysozyme, and 50 ~g/ml NaN3), and allowed to incubate for 5 minutes at 25 C. To this preparation was added 5 ml of extraction buffer-2 (1.5 M NaCl, 100 WO94/14973 PCT~S93/12C26 mM CaCl2, 100 mM MgCl2, 0.02 mg/ml DNase 1, and 50 ~g/ml - ovomucoid protease inhibitor), and this suspension was allowed to incubate for 5 minutes at 25 C. Cells were further lysed by sonication for 4 cycles of 30 seconds at 70 W on a Branson Sonic power sonifier. Insoluble cellular debris was separated by centrifugation at 25,000 g for 60 minutes at 4-C. The cell lysate contAi~;ng CDllb I-domain fusion peptide was stored at -70 C.
10Monoclonal antibody/CDllb I-domain fusion protein complexes were formed by inCl~h~ting the cell lysate prepared as described above with a monoclonal antibody that r~co~ni7ed the FLAG peptide of the CDllb I-domain fusion protein (anti-FLAG Ml; International Biotechnologies~ Inc.). These complexes were i~c~lh~ted with '25I-rNIF and precipitated with protein A-sepharose (Calbiochem). One mi~,G~.am of Ml monoclonal antibody was incubated with 390 ~1 of the cell lysate that contained CDllb I-domain fusion peptide, in the presence of 105 cpm '25I-rNIF (specific activity 47.5 ~Ci/~g; see Example 14(B)). The total reaction volume was 400 ~1, and the reaction proceeded for 18 hours at 4 C. Immune complexes were precipitated with 100 ~1 protein A-Sepharose (Pharmacia). The protein A-Sepharose was prepared as a 1:1 slurry in TACTS 20 bu~fer and preblocked with 0.5% BSA. After precipitation, Sepharose beads were washed three times with TACTS 20 W094/14973 PCT~S93/12626 ?S99 lSo /1 buffer, and immune complexes were released by the addition of Tris-glycine sample buffer (Novex) under reducing conditions (100 mM dithiothreitol).
Precipitated, labeled proteins were resolved by 4-20%
gradient SDS-PAGE (Novex) and visualized by autoradiography.
The l~'I-rNIF was precipitated by M1 monoclonal antibody in the presence of cell lysate that contained recombinant I-domain peptide. In contrast, I~I-rNIF was not precipitated in the absence of this cell lysate or when the M1 monoclonal antibody was substituted with a monoclonal antibody that did not react with the the FLAG
portion of the fusion protein (anti-gpIIbIIIa).
Moreover, Ml did not precipitate I~I-rNIF in the presence of another fusion protein comprising FLAG and bacterial alkaline phosphatase (pFLAG-BAP; International Biotechnologies, Inc.). These data demonstrate direct and specific interaction between NIF and the I-domain peptide of CDllb/CD18.

Claims (210)

Claims

1. A Neutrophil Inhibitory Factor comprising an amino acid sequence selected from the group consisting of (a) Arg-X1-X2-Phe-Leu-X3-X4-His-Asn-Gly-Tyr-Arg-Ser-X5-Leu-Ala-Leu-Gly-His-X6-X7-Ile, wherein X1 is Leu or Met; X5 is Lys, Arg, Leu or Ile; X6 is Val or Ile; and X7 is Ser, Gly or Asn;
(b) Ala-X8-X9-Ala-Ser-X10-Met-Arg-X11-Leu-X12-Tyr-Asp-Cys-X13-Ala-Glu-X14-Ser-Ala-Tyr-X15-Ser-Ala, wherein X8 is His or Pro; X9 is Thr, Arg or Ser; X10 is Arg or Lys;
X11 is Ile or Tyr; X12 is Asp, Lys or Glu; X13 is Asp or Glu; X14 is Gly, Lys or Arg; and X15 is Glu, Met, Thr or Val;
(c) Ser-X16-Phe-Ala-Asn-X17-Ala-Trp-Asp-X18-Arg-Glu-Lys-X19-Gly-Cys-Ala-Val-Val-X20-Cys, wherein X16 is Asn or Asp; X17 is Val or Leu; X18 is Ala or Thr; X19 is Leu, Val or Phe; and X20 is Thr, Lys or Asn;
(d) His-Val-Val-Cys-His-X21-X22-Pro-Lys, wherein X21 is Tyr or Ile; X22 is Gly or no residue;
(e) Ile-Tyr-X23-X24-Gly-X25-Pro-Cys-X26-X27-Cys-X28-X29-Tyr, wherein X23 is Thr, Ser, Lys or Glu; X24 is Thr, Val or Ile; X25 is Val, Lys or Thr; X26 is Arg, Ser or Asp; X27 is Asn, Gly, Asp or Arg; X28 is Asn, Ser or Thr;
and X29 is Gly, Glu or Asp; and (f) Cys-X30-X31-Asp-X32-Gly-Val-Cys-X33-Ile, wherein X30 is His, Ile or Asn; X31 is Ala, Pro or Asp; X32 is Glu, Val, Asp or Ile; X33 is Ile, Val or Phe.

2. A Neutrophil Inhibitory Factor of claim 1, further characterized as having neutrophil inhibitory activity.

3. A Neutrophil Inhibitory Factor of claim 2, wherein said neutrophil inhibiting activity is demonstrated by an assay selected from the group consisting of assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.

4. A Neutrophil Inhibitory Factor of claim 3, further characterized as having an IC50 for inhibiting neutrophil activity of about 500 nM or less.

5. A Neutrophil Inhibitory Factor of claim 4, further characterized by its ability to bind to the integrin complex, CD11b/CD18.

6. A Neutrophil Inhibitory Factor of Claim 4 further characterized by its ability to bind to a recombinant peptide comprising the I-domain of the integrin complex, CD11b/CD18.

7. A Neutrophil Inhibitory Factor of claim 1, further characterized as having eosinophil inhibiting activity.

8. A Neutrophil Inhibitory Factor of claim 7, wherein said eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells.

9. A Neutrophil Inhibitory Factor of claim 8, further characterized as having an IC50 for inhibiting eosinophil activity of about 500 nM or less.

10. A Neutrophil Inhibitory Factor comprising the amino acid sequence shown in Figure 8.

11. A Neutrophil Inhibitory Factor comprising an amino acid sequence selected from the group consisting of the amino acid sequences shown in Figure 9 for 3P, 1P, 2FL, 3FL, 4FL and 6FL.

12. A Neutrophil Inhibitory Factor comprising an amino acid sequence selected from the group consisting of the amino acid sequences shown in Figure 16 for PCR-NIF#7, AcaNIF19 and AcaNIF24.

13. A Neutrophil Inhibitory Factor comprising an amino acid sequence selected from the group consisting of the amino acid sequences shown in Figure 16 for PCR-NIF#20, AcaNIF4, AcaNIF6, AcaNIF7, AcaNIF9, AceNIF1.

14. A mutant Neutrophil Inhibitory Factor comprising the amino acid sequence shown in Figure 8, wherein one or more of asparagine residues at positions 10, 18, 87, 110, 130, 197 or 223 is replaced by an glutamine residue.

15. A mutant Neutrophil Inhibitory Factor of claim 14, further characterized as having neutrophil inhibitory activity.

16. A mutant Neutrophil Inhibitory Factor of claim 15, wherein said neutrophil inhibiting activity is demonstrated by an assay selected from the group consisting of assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.

17. A mutant Neutrophil Inhibitory Factor of claim 16, further characterized as having an IC50 for inhibiting neutrophil activity of about 500 nM or less.

18. A mutant Neutrophil Inhibitory Factor of claim 17, further characterized by its ability to bind to the integrin complex, CD11b/CD18.

19. A Neutrophil Inhibitory Factor of Claim 17 further characterized by its ability to bind to a recombinant peptide comprising the I-domain of the integrin complex, CD11b/CD18.

20. A mutant Neutrophil Inhibitory Factor of claim 14, further characterized as having eosinophil inhibiting activity.

21. A mutant Neutrophil Inhibitory Factor of claim 20, wherein said eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells.

22. A mutant Neutrophil Inhibitory Factor of claim 21, further characterized as having an IC50 for inhibiting eosinophil activity of about 500 nM or less.

23. A Neutrophil Inhibitory Factor comprising an amino acid sequence which is encoded by a nucleic acid sequence which is sufficiently complementary to hybridize to the probe having a sequence selected from the group consisting of 5-CTCGAATTCT(GATC)GC(ATC)AT(ATC)(CT)T(GATC)GG(ATC)TGGGC-3' and 5'-CTCGAATTCTT(TC)TCTGG(GA)AA(GA)CG(GA)TC(GA)AA-3'.

24. A Neutrophil Inhibitory Factor of claim 23, further characterized as having neutrophil inhibitory activity.

25. A Neutrophil Inhibitory Factor of claim 24, wherein said neutrophil inhibiting activity is demonstrated by an assay selected from the group consisting of assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.

26. A Neutrophil Inhibitory Factor of claim 25, further characterized as having an IC50 for inhibiting neutrophil activity of about 500 nM or less.

27. A Neutrophil Inhibitory Factor of claim 26, further characterized by its ability to bind to the integrin complex, CD11b/CD18.

28. A Neutrophil Inhibitory Factor of Claim 26 furthr characterized by its ability to bind to a recombinant peptide comprising the I-domain of the integrin, CD11b/CD18.

29. A Neutrophil Inhibitory Factor of claim 23, further characterized as having eosinophil inhibiting activity.

30. A Neutrophil Inhibitory Factor of claim 29, wherein said eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells.

31. A Neutrophil Inhibitory Factor of claim 30, further characterized as having an IC50 for inhibiting eosinophil activity of about 500 nM or less.

32. A DNA probe having the sequence of 5'-CTCGAATTCT(GATC)GC(ATC)AT(ATC)(CT)T(GATC)GG(ATC)TGGGC
-3'.

33. A DNA probe having the sequence of 5'-CTCGAATTCTT(TC)TCTGG(GA)AA(GA)CG(GA)TC(GA)AA-3'.

34. A Neutrophil Inhibitory Factor comprising an amino acid sequence which is encoded by a nucleic acid sequence which is sufficiently complementary to hybridize to a probe having at least about 12 nucleotides which is complementary to a portion of the sequence of Figure 8.

35. A Neutrophil Inhibitory Factor of claim 34, further characterized as having neutrophil inhibitory activity.

36. A Neutrophil Inhibitory Factor of claim 35, wherein said neutrophil inhibiting activity is demonstrated by an assay selected from the group consisting of assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.

37. A Neutrophil Inhibitory Factor of claim 36, further characterized as having an IC50 for inhibiting neutrophil activity of about 500 nM or less.

38. A Neutrophil Inhibitory Factor of claim 37, further characterized by its ability to bind to the integrin complex, CD11b/CD18.

39. A Neutrophil Inhibitory Factor of Claim 37 further characterized by its ability to bind to a recombinant peptide comprising the I-domain of the integrin complex, CD11b/CD18.

40. A Neutrophil Inhibitory Factor of claim 34, further characterized as having eosinophil inhibiting activity.

41. A Neutrophil Inhibitory Factor of claim 40, wherein said eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells.

42. A Neutrophil Inhibitory Factor of claim 41, further characterized as having an IC50 for inhibiting eosinophil activity of about 500 nM or less.

43. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid sequence for a Neutrophil Inhibitory Factor.

44. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid sequence for a Neutrophil Inhibitory Factor which is selected from the group consisting of a Neutrophil Inhibitory Factor of claim 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 34, 35, 36, 37, 38, 40, 41 or 42.

45. An isolated nucleic acid molecule of claim 44, wherein said nucleic acid molecule is a DNA molecule.

46. An expression vector comprising an isolated nucleic acid molecule of claim 45 operably linked to control sequences recognized by a host cell transformed with the vector.

47. An expression vector of claim 46 selected from the group consisting of pHIL7SP-N1c10, pSG5/NIF1FLCR1, pMa5-NI1/3 pAN-NIF-1FL, pYAM7SP-hNIF1/.DELTA.,G11-5, pYAM7SP-hNIF1/.DELTA.,G11-5, pYAMSP-AcaNIF4, pYAMSP-AcaNIF6, pYAMSP-AcaNIF9, pYAMSP-AcaNIF24 and pAN-AceNIF3.

48. A host cell transformed with an expression vector of claim 47.

49. A method for making a biologically active Neutrophil Inhibitory Factor comprising the step of culturing host cells having an expression vector which encodes a gene for a Neutrophil Inhibitory Factor inserted in said cells under cell culture conditions whereby said Neutrophil Inhibitory Factor is expressed.

50. A method of claim 49, wherein said gene is obtained from a parasitic worm.

51. A method of claim 50, wherein said parasitic worm is selected from the group consisting of the species of Platyhelminthes, Nematoda, Nematomorpha and Acanthocephala.

52. A method of claim 51, wherein said parasitic worm is selected from the group consisting of the species of Nematoda.

53. A method of claim 52, wherein said species is a canine hookworm.

54. A method of claim 53, wherein said canine hookworm is a Ancylostoma caninum.

55. A method of claim 53, wherein said canine hookworm is a Ancylostoma ceylanicum.

56. A method of claim 52, wherein said species is a canine roundworm.

57. A method of claim 56, wherein said canine roundworm is Toxocara canis.

58. A method of claim 49, wherein said host cell is selected from the group consisting of bacterial cell, yeast cell, mammalian cell and insect cell.

59. A method of claim 58, wherein said host cell is a bacterial cell.

60. A method of claim 59, wherein said bacterial cell is E. coli. WK6.

61. A method of claim 58, wherein said host cell is a yeast cell.

62. A method of claim 61, wherein said yeast cell is Saccharomyces cerevisiae.

63. A method of claim 61, wherein said yeast cell is Pichia pastoris.

64. A method of claim 58, wherein said host cell is a mammalian cell.

65. A method of claim 64, wherein said mammalian cell is COS-7.

66. A method of claim 64, wherein said mammalian cell is CHO-K1.

67. A method of making biologically active Neutrophil Inhibitory Factor comprising the step of culturing host cells having an expression vector which encodes a gene for a Neutrophil Inhibitory Factor inserted in said cells under cell culture conditions.
Whereby said Neutrophil Inhibitory Factor is expressed, wherein said gene encodes for the Neutrophil Inhibitory Factor of claim 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 34, 35, 36, 37, 38, 40, 41 or 42.

68. A method of claim 67, wherein said host cell is E. coli. WK6.

69. A method of claim 67, wherein said host cell is Pichia pastoris.

70. A method of claim 67, wherein said host cell is CHO-K1.

71. A Neutrophil Inhibitory Factor made by the method of claim 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66.

72. A Neutrophil Inhibitory Factor made by the method of claim 67.

73. A Neutrophil Inhibitory Factor made by the method of claim 68.

74. A Neutrophil Inhibitory Factor made by the method of claim 69.

75. A Neutrophil Inhibitory Factor made by the method of claim 70.

76. A method for making a biologically active Neutrophil Inhibitory Factor comprising the step of culturing host cells having an expression vector which encodes a gene for a Neutrophil Inhibitory Factor inserted in said cells under cell culture conditions whereby Neutrophil Inhibitory Factor is secreted.

77. A method of claim 76, wherein said gene is obtained from a parasitic worm.

78. A method of claim 77, wherein said parasitic worm is selected from the group consisting of the species of Platyhelminthes, Nematoda, Nematomorpha and Acanthocephala.

79. A method of claim 78, wherein said parasitic worm is selected from the group consisting of the species of Nematoda.

80. A method of claim 79, wherein said species is a canine hookworm.

81. A method of claim 80, wherein said canine hookworm is a Ancylostoma caninum.

82. A method of claim 80, wherein said canine hookworm is a Ancylostoma ceylanicum.

83. A method of claim 79, wherein said species is a canine roundworm.

84. A method of claim 83, wherein said canine roundworm is Toxocara canis.

85. A method of claim 76, wherein said host cell is selected from the group consisting of bacterial cell, yeast cell, mammalian cell and insect cell.

86. A method of claim 85, wherein said host cell is a bacterial cell.

87. A method of claim 86, wherein said bacterial cell is E. coli. WK6.

88. A method of claim 85, wherein said host cell is a yeast cell.

89. A method of claim 88, wherein said yeast cell is Saccharomyces cerevisiae.

90. A method of claim 88, wherein said yeast cell is Pichia pastoris.

91. A method of claim 85, wherein said host cell is a mammalian cell.

92. A method of claim 91, wherein said mammalian cell is COS-7.

93. A method of claim 91, wherein said mammalian cell is CHO-K1.

94. A method of making biologically active Neutrophil Inhibitory Factor comprising the step of culturing host cells having an expression vector which encodes a gene for Neutrophil Inhibitory Factor inserted in said cells under cell culture conditions whereby Neutrophil Inhibitory Factor is secreted, wherein said gene encodes for the protein of claim 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 34, 35, 36, 37, 38, 40, 41 or 42.

95. A method of claim 94, wherein said host cell is E. coli. WK6.

96. A method of claim 94, wherein said host cell is Pichia pastoris.

97. A method of claim 94, wherein said host cell is CHO-K1.

98. A Neutrophil Inhibitory Factor made by the method of claim 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93.

99. A Neutrophil Inhibitory Factor made by the method of claim 94.

100. A Neutrophil Inhibitory Factor made by the method of claim 95.

101. A Neutrophil Inhibitory Factor made by the method of claim 96.

102. A Neutrophil Inhibitory Factor made by the method of claim 97.

103. A method of making Neutrophil Inhibitory Factor comprising the steps of preparing cDNA library from a source suspected of containing Neutrophil Inhibitory Factor and hybridizing oligonucleotide probes sufficiently complementary to hybridize to a nucleic acid encoding a Neutrophil Inhibitory Factor to the cDNA
library.

104. A method of claim 103, wherein said oligonucleotide probes have the sequences 5'-CTCGAATTCT(GATC)GC(ATC)AT(ATC)(CT)T(GATC)GG(ATC)TGGGC
-3' and 5'-CTCGAATTCTT(TC)TCTGG(GA)AA(GA)CG(GA)TC(GA)AA-3'.

105. A method of claim 104, further comprising demonstrating that said Neutrophil Inhibitory Factor has neutrophil inhibiting activity.

106. A method of claim 105, wherein said neutrophil inhibiting activity is demonstrated by an assay selected from the group consisting of assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.

107. A method of claim 106, wherein said Neutrophil Inhibitory Factor is further characterized as having an IC50 for inhibiting neutrophil activity of about 500 nM
or less.

108. A method of claim 107, further comprising demonstrating that said Neutrophil Inhibitory Factor has ability to bind to the integrin complex, CD11b/CD18.

109. A method of claim 107, further comprising demonstrating the said Neutrophil Inhibitory Factor has ability to bind to a recombilant peptide comprising the I-domain of the integrin complex CD11b/CD18.

110. A method of claim 104, further comprising demonstrating that said Neutrophil Inhibitory Factor has eosinophil inhibiting activity.

111. A method of claim 110, wherein said eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells.

112. A method of claim 111, wherein said Neutrophil Inhibitory Factor is further characterized as having an IC50 for inhibiting eosinophil activity of about 500 nM
or less.

113. A Neutrophil Inhibitory Factor made by the method of claim 103, 104, 105, 106, 107, 108, 110, 111, or 112.

114. A method of detecting in a sample the presence of a nucleic acid molecule encoding a Neutrophil Inhibitory Factor comprising the steps of (1) combining a solution of said sample with a nucleic acid probe that is sufficiently complementary to hybridize to a nucleic acid encoding a Neutrophil Inhibitory Factor and (2) detecting the presence of said probe.

115. A method of detecting in a sample the presence of a nucleic acid molecule encoding a Neutrophil Inhibitory Factor comprising the steps of (1) combining a solution of said sample with a nucleic acid probe that is sufficiently complementary to hydridize to a nucleic acid encoding a Neutrophil Inhibitory Factor and (2) detecting the presence of said probe, wherein said probe hybridizes with a Neutrophil Inhibitory Factor is selected from a group consisting of the Neutrophil Inhibitory Factors of claim 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12 and 13.

116. A method of claim 115, wherein said Neutrophil Inhibitory Factor is a Neutrophil Inhibitory Factor comprising the amino acid sequence shown in Figure 8.

117. A method of claim 114, wherein said probe is a oligonucleotide probe selected from the group consisting of 5'-CTCGAATTCT(GATC)GC(ATC)AT(ATC)(CT)T(GATC)GG(ATC)TGGGC
-3' and 5'-CTCGAATTCTT(TC)TCTGG(GA)AA(GA)CG(GA)TC(GA)AA-3'.

118. A monoclonal antibody which is capable of binding a Neutrophil Inhibitory Factor.

119. A monoclonal antibody which is capable of binding a Neutrophil Inhibitory Factor which is selected from the group consisting of the Neutrophil Inhibitory Factors of claim 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21 and 22.

120. A monoclonal antibody which is capable of binding Neutrophil Inhibitory Factor wherein said Neutrophil Inhibitory Factor is a Neutrophil Inhibitory Factor of Figure 8.

121. A monoclonal antibody of claim 120, wherein said monoclonal antibody is further characterized as an IgG.

122. A monoclonal antibody of claim 121, wherein said monoclonal antibody is further characterized as binding to the same epitope on said Neutrophil Inhibitory Factor as is bound by the monoclonal antibody, 3D2.

123. A monoclonal antibody of claim 121, wherein said monoclonal antibody is 3D2.

124. A hybridoma secreting a monoclonal antibody of claim 122 or 123.

125. A method of isolating Neutrophil Inhibitory Factor comprising contacting a sample thought to contain Neutrophil Inhibitory Factor with a monoclonal antibody which is capable of binding to said Neutrophil Inhibitory Factor.

126. A method of claim 125, wherein said monoclonal antibody is further characterized as binding to the same epitope on said Neutrophil Inhibitory Factor as is bound by the monoclonal antibody, 3D2.

127. A method of claim 125, wherein said monoclonal antibody is 3D2.

128. A method of claim 126 or 127, wherein said monoclonal antibody is covalently attached to a chromatographic resin.

129. A method of claim 128, wherein said chromatographic resin is Emphaze Biosupport Medium.

130. A method of detecting Neutrophil Inhibitory Factor in a sample comprising contacting said sample with a monoclonal antibody which is capable of binding to said Neutrophil Inhibitory Factor.

131. A method of claim 130, wherein said monoclonal antibody is immobilized onto a plastic surface.

132. A method of claim 131, wherein said plastic is polystyrene.

133. A method of claim 132, wherein said immobilization of monoclonal antibody is achieved by passive absorption.

134. A method of claim 133, further comprising simultaneously contacting said monoclonal antibody, sample and a Neutrophil Inhibitory Factor which has been which has been covalently linked to a detectable label.

135. A method of claim 133, further comprising first contacting said monoclonal antibody with said sample, then contacting said monoclonal antibody with a Neutrophil Inhibitory Factor of claim 9 which has been covalently linked to a detectable label.

136. A method of claim 135, wherein said detectable label is radioisotope or enzyme.

137. A method of claim 136, wherein said detectable label is selected from the group consisting of iodine-125, alkaline phosphatase, .beta.-galactosidase and horseradish peroxidase.

138. A method of claim 137, wherein said label is iodine-125.

139. A method of claim 138, wherein said monoclonal antibody is further characterized as binding to the same epitope on a Neutrophil Inhibitory Factor of claim 9 as is bound by the monoclonal antibody 3D2.

140. A method of claim 138, wherein said monoclonal antibody is is further characterized as being 3D2.

141. A method of detecting in a sample the presence of a Neutrophil Inhibitory Factor mimic which competes with Neutrophil Inhibitory Factor for binding to CD11b/CD18 receptor comprising contacting said sample with CD11b/CD18 receptor.

142. A method of claim 141, wherein said Neutrophil Inhibitory Factor mimic is selected from the group consisting of small molecules, peptides, peptide analogs and proteins.

143. A method of claim 142, wherein said CD11b/CD18 receptor is bound to anti-CD11b/CD18 monoclonal antibody which has been immobilized onto a plastic surface.

144. A method of claim 143, wherein said immobilization of said monoclonal antibody onto said plastic surface is achieved by passive absorption.

145. A method of claim 144, wherein said monoclonal antibody is LM2.

146. A method of claim 145, wherein said plastic is polystyrene.

147. A method of claim 146, further comprising simultaneously contacting said CD11b/CD18 receptor, said sample and a Neutrophil Inhibitory Factor which is capable of being linked to a detectable label.

148. A method of claim 147, wherein said Neutrophil Inhibitory Factor is further characterized as being covalently attached to biotin.

149. A method of claim 148, wherein said detectable label is further characterized as being covalently attached to avidin.

150. A method of claim 149, wherein said detectable label is an enzyme.

151. A method of claim 150, wherein said enzyme is selected from a group consisting of alkaline phosphatase, .beta.-galactosidase and horseradish peroxidase.

152. A method of claim 151, wherein said enzyme is alkaline phosphatase.

153. A Neutrophil Inhibitory Factor mimic identified by the method of claim 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 or 152 having neutrophil inhibitory activity.

154. A Neutrophil Inhibitory Factor mimic of claim 153, wherein said neutrophil inhibiting activity is demonstrated by an assay selected from the group consisting of assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.

155. A Neutrophil Inhibitory Factor mimic of claim 154, further characterized as having an IC50 for inhibiting neutrophil activity of about 500 nM or less.

156. A Neutrophil Inhibitory Factor mimic identified by the method of claim 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 or 152 having eosinophil inhibitory activity.

157. A Neutrophil Inhibitory Factor mimic of claim 156, wherein said eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells.

158. A Neutrophil Inhibitory Factor mimic of claim 157, further characterized as having an IC50 for inhibiting eosinophil activity of about 500 nM or less.

159. A method of detecting in a sample the presence of a Neutrophil Inhibitory Factor antagonist which prevents Neutrophil Inhibitory Factor from binding to CD11b/CD18 receptor comprising contacting said sample with CD11b/CD18 receptor.

160. A method of claim 159, wherein said Neutrophil Inhibitory Factor antagonist is selected from the group consisting of small molecules, peptides, peptide analogs and proteins.

161. A method of claim 160, wherein said CD11b/CD18 receptor is bound to anti-CD11b/CD18 monoclonal antibody which has been immobilized onto a plastic surface.

162. A method of claim 161, wherein said immobilization of said monoclonal antibody onto said plastic surface is achieved by passive absorption.

163. A method of claim 162, wherein said monoclonal antibody is LM2.

164. A method of claim 163, wherein said plastic is polystyrene.

165. A method of claim 164, further comprising simultaneously contacting said CD11b/CD18 receptor, said sample and a Neutrophil Inhibitory Factor which is capable of being linked to a detectable label.

166. A method of claim 165, wherein said Neutrophil Inhibitory Factor is further characterized as being covalently attached to biotin.

167. A method of claim 166, wherein said detectable label is further characterized as being covalently attached to avidin.

168. A method of claim 167, wherein said detectable label is an enzyme.

169. A method of claim 168, wherein said enzyme is selected from a group consisting of alkaline phosphatase, .beta.-galactosidase and horseradish peroxidase.

170. A method of claim 169, wherein said enzyme is alkaline phosphatase.

171. A Neutrophil Inhibitory Factor antagonist identified by the method of claim 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170 and lacking neutrophil inhibitory activity.

172. A Neutrophil Inhibitory Factor antagonist of claim 171, wherein said neutrophil inhibiting activity is demonstrated by an assay selected from the group consisting of assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.

173. A Neutrophil Inhibitory Factor antagonist of claim 172, further characterized as having an IC50 for inhibiting neutrophil activity of about 1000 nM to about 1 mM.

174. A Neutrophil Inhibitory Factor antagonist identified by the method of claim 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170 and lacking eosinophil inhibitory activity.

175. A Neutrophil Inhibitory Factor antagonist of claim 174, wherein said eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells.

176. A Neutrophil Inhibitory Factor antagonist of claim 175, further characterized as having an IC50 for inhibiting eosinophil activity of about 1000 nM to about 1 mM.

177. A method of treating in a mammal an inflammatory condition characterized by abnormal neutrophil activation comprising administering to said mammal a therapeutically effective amount of a Neutrophil Inhibitory Factor.

178. A method of treating in a mammal an inflammatory condition characterized by abnormal neutrophil activation comprising administering to said mammal a therapeutically effective amount of a Neutrophil Inhibitory Factor, wherein said Neutrophil Inhibitory Factor is selected from the group consisting of the Neutrophil Inhibitory Factors of claim 1, 4, 9, 10, 11, 12, 13, 14, 17, 23, 26, 34, 37 or 49.

179. A method of preventing in a mammal an inflammatory condition characterized by abnormal eosinophil activation comprising administering to said mammal a therapeutically effective amount of a Neutrophil Inhibitory Factor.

180. A method of preventing in a mammal an inflammatory condition characterized by abnormal eosinophil activation comprising administering to said mammal a therapeutically effective amount of a Neutrophil Inhibitory Factor, wherein said Neutrophil Inhibitory Factor is selected from the group consisting of the Neutrophil Inhibitory Factors of claim 1, 8, 9, 10, 11, 12, 13, 14, 22, 23, 31, 34, 42 or 49.

181. A method of detecting in a sample the presence of a Neutrophil Inhibitory Factor mimic which competes with Neutrophil Inhibitory Factor for binding to a recombinant peptide comprising the I-domain of the CD11b/CD18 receptor, comprising contacting said sample with a recombinant peptide comprising the I-domain of the CD11b/CD18 receptor.

182. A method of claim 181, wherein said Neutrophil Inhibitory Factor mimic is selected from the group consisting of small molecules, peptides, peptide analogs and proteins.

183. A method of claim 182, further comprising simultaneously contacting said recombinant peptide, said sample and a Neutrophil Inhibitory Factor which is capable of being linked to a detectable label.

184. A method of claim 183, wherein said detectable label is a 125I.

185. A method of claim 183, wherein said Neutrophil Inhibitory Factor is further characterized as being covalently attached to biotin.

186. A method of claim 185, wherein said detectable label is further characterized as being covalently attached to avidin.

187. A method of claim 186, wherein said detectable label is an enzyme.

188. A method of claim 187, wherein said enzyme is selected from a group consisting of alkaline phosphatase, .beta.-galactosidase and horseradish peroxidase.

189. A method of claim 188, wherein said enzyme is alkaline phosphatase.

190. A Neutrophil Inhibitory Factor mimic identified by the method of claim 181, 182, 183, 184, 185, 186, 187, 188 or 189 having neutrophil inhibitory activity.

191. A Neutrophil Inhibitory Factor mimic of claim 190, wherein said neutrophil inhibiting activity is demonstrated by an assay selected from the group consisting of assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.

192. A Neutrophil Inhibitory Factor mimic of claim 191, further characterized as having an IC50 for inhibiting neutrophil activity of about 500 nM or less.

193. A Neutrophil Inhibitory Factor mimic identified by the method of claim 181, 182, 183, 184, 185, 186, 187, 188 or 189 having eosinophil inhibitory activity.

194. A Neutrophil Inhibitory Factor mimic of claim 193, wherein said eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells.

195. A Neutrophil Inhibitory Factor mimic of claim 194, further characterized as having an IC50 for inhibiting eosinophil activity of about 500 nM or less.

196. A method of detecting in a sample the presence of a Neutrophil Inhibitory Factor antagonist which prevents Neutrophil Inhibitory Factor from binding to a recombinant peptide comprising the I-domain of the CD11b/CD18 receptor, comprising contacting said sample with a recombinant peptide comprising the I-domain of the CD11b/CD18 receptor.

197. A method of claim 196, wherein said Neutrophil Inhibitory Factor antagonist is selected from the group consisting of small molecules, peptides, peptide analogs and proteins.

198. A method of claim 164, further comprising simultaneously contacting said recombinant peptide, said sample and a Neutrophil Inhibitory Factor which is capable of being linked to a detectable label.

199. A method of claim 198, wherein said detectable label is a 125I.

200. A method of claim 198, wherein said Neutrophil Inhibitory Factor is further characterized as being covalently attached to biotin.

201. A method of claim 200, wherein said detectable label is further characterized as being covalently attached to avidin.

202. A method of claim 201, wherein said detectable label is an enzyme.

203. A method of claim 202, wherein said enzyme is selected from a group consisting of alkaline phosphatase, .beta.-galactosidase and horseradish peroxidase.

204. A method of claim 203, wherein said enzyme is alkaline phosphatase.

205. A Neutrophil Inhibitory Factor antagonist identified by the method of claim 196, 197, 198, 199, 200, 201, 202, 203 or 204 having neutrophil inhibitory activity.

206. A Neutrophil Inhibitory Factor antagonist of claim 205, wherein said neutrophil inhibiting activity is demonstrated by an assay selected from the group consisting of assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.

207. A Neutrophil Inhibitory Factor antagonist of claim 206, further characterized as having an IC50 for inhibiting neutrophil activity of about 1000 nM to about 1 mM.

208. A Neutrophil Inhibitory Factor antagonist identified by the method of claim 196, 197, 198, 199, 200, 201, 202, 203 or 204 and lacking eosinophil inhibitory activity.

209. A Neutrophil Inhibitory Facto antagonist of claim 208, wherein said eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells.

210. A Neutrophil Inhibitory Factor antagonist of claim 209, further characterized as having an IC50 for inhibiting eosinophil activity of about 1000 nM to about 1 mM.