Compositions comprising complement receptor type 1 molecules having carbohydrate structures that are selectin ligands

The present invention provides compositions comprising at least one complement moiety and at least one carbohydrate moiety, and methods of producing such compositions. In particular, the compositions of the invention comprise complement proteins related to the complement receptor type 1, and further comprise ligands for intercellular molecules, such as selectins. In a preferred embodiment, the compositions comprise a complement-related protein in combination with the Lewis X antigen or the sialyl Lewis X antigen. The compositions of the invention have use in the diagnosis or therapy of disorders involving complement activity and inflammation. Pharmaceutical compositions are also provided for treating or reducing inflammation mediated by inappropriate complement activity and intercellular adhesion.

1. FIELD OF THE INVENTION
 In its broadest aspect, the present invention provides compositions
 comprising at least one complement moiety and at least one carbohydrate
 moiety, and methods of producing such compositions. In particular, the
 compositions of the invention comprise complement proteins related to the
 complement receptor type 1, and further comprise ligands for intercellular
 adhesion molecules, such as selectins. In a preferred embodiment, the
 compositions comprise a complement receptor type 1, or fragment or
 derivative thereof, in combination with the Lewis X antigen or the sialyl
 Lewis X antigen. The compositions of the invention have use in the
 diagnosis or therapy of disorders involving complement activity and
 inflammation. Pharmaceutical compositions are also provided for treating
 or reducing inflammation mediated by inappropriate complement activity and
 intercellular adhesion.
 2. BACKGROUND OF THE INVENTION
 2.1. THE COMPLEMENT SYSTEM
 The complement system is a group of proteins that constitute about 10
 percent of the globulins in the normal serum of humans (Hood, L. E., et
 al., 1984, Immunology, 2d Ed., The Benjamin/Cummings Publishing Co., Menlo
 Park, Calif., p. 339). Complement (C) plays an important role in the
 mediation of immune and allergic reactions (Rapp, H. J. and Borsos, T,
 1970, Molecular Basis of Complement Action, Appleton-Century-Crofts
 (Meredity), New York). The activation of complement components leads to
 the generation of a group of factors, including chemotactic peptides that
 mediate the inflammation associated with complement dependent diseases.
 The sequential activation of the complement cascade may occur via the
 classical pathway involving antigen-antibody complexes, or by the
 alternative pathway which involves the recognition of foreign structures
 such as, certain cell wall polysaccharides. The activities mediated by
 activated complement proteins include lysis of target cells, chemotaxis,
 opsonization, stimulation of vascular and other smooth muscle cells, and
 functional aberrations such as degranulation of mast cells, increased
 permeability of small blood vessels, directed migration of leukocytes, and
 activation of B lymphocytes and macrophages (Eisen, H. N., 1974,
 Immunology, Harper & Row Publishers, Inc. Hagerstown, Md., p. 512).
 During proteolytic cascade steps, biologically active peptide fragments,
 the anaphylatoxins C3a, C4a, and C5a (See WHO Scientific Group, 1977, WHO
 Tech Rep. Ser. 606:5 and references cited therein), are released from the
 third (C3), fourth (C4), and fifth (C5) native complement components
 (Hugli, T. E., 1981, CRC Crit. Rev. Immunol. 1:321; Bult, H. and Herman,
 A. G., 1983, Agents Actions 13:405).
 2.2. COMPLEMENT RECEPTORS
 COMPLEMENT RECEPTOR 1 (CR1). The human C3b/C4b receptor, termed CR1 or
 CD35, is present on erythrocytes, monocytes/macrophages, granulocytes, B
 cells, some T cells, splenic follicular dendritic cells, and glomerular
 podocytes (Fearon D. T., 1980, J. Exp. Med. 152:20, Wilson, J. G., et al.,
 1983, J. Immunol. 131:684; Reynes, M., et al., 1976 N. Engl. J. Med.
 295:10; Kazatchkine, M. D., et al., 1982, Clin. Immunol. Immunopathol.
 27:210). CR1 specifically binds C3b, C4b and iC3b.
 CR1 can inhibit the classical and alternative pathway C3/C5 convertases and
 act as a cofactor for the cleavage of C3b and C4b by factor I, indicating
 that CR1 also has complement regulatory functions in addition to serving
 as a receptor (Fearon, D. T., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:5867;
 Iida, K. I. and Nussenzweig, V., 1981, J. Exp. Med. 153:1138). In the
 alternative pathway of complement activation, the bimolecular complex
 C3b,Bb is a C3 enzyme (convertase). CR1 (and factor H, at higher
 concentrations) can bind to C3b and can also promote the dissociation of
 C3b,Bb. Furthermore, formation of C3b,CR1 (and C3b,H) renders C3b
 susceptible to irreversible proteolytic inactivation by factor I,
 resulting in the formation of inactivated C3b (iC3b). In the classical
 pathway of complement activation, the complex C4b,2a is the C3 convertase.
 CR1 (and C4 binding protein, C4bp, at higher concentrations) can bind to
 C4b, and can also promote the dissociation of C4b,2a. The binding renders
 C4b susceptible to irreversible proteolytic inactivation by factor I
 through cleavage to C4c and C4d (inactivated complement proteins).
 CR1 has been shown to have homology to complement receptor type 2 (CR2)
 (Weis, J.J., et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:5639-5643).
 CR1 is a glycoprotein comprising multiple short consensus repeats (SCRs)
 arranged in 4 long homologous repeats (LHRs). The most C-terminal LHR
 called LHR-D is followed by 2 additional SCRs, a transmembrane region and
 a cytoplasmic region (Klickstein, et al., 1987, J. Exp. Med., 165:1095;
 Klickstein, et al. 1988, J. Exp. Med., 168:1699-1717). Erythrocyte CR1
 appears to be involved in the removal of circulating immune complexes in
 autoimmune patients and its levels may correlate with the development of
 AIDS (Inada, et al., 1986, AIDS Res. 2:235; Inada, et al., 1989, Ann.
 Rheu. Dis. 4:287).
 Four allotypic forms of CR1 have been found, differing by increments of
 40,000-50,000 daltons molecular weight. The two most common forms, the F
 and S allotypes, also termed the A and B allotypes, have molecular weights
 of 250,000 and 290,000 daltons respectively, (Dykman, T. R., et al., 1983,
 Proc. Natl. Acad. Sci. U.S.A. 80:1698; Wong, W. W., et al., 1983, J. Clin.
 Invest. 72:685), and two rarer forms have molecular weights of 210,000 and
 290,000 daltons (Dykman, T. R., et al., 1984, J. Exp. Med. 159:691;
 Dykman, T. R., et al., 1985, J. Immunol. 134:1787). These differences
 apparently represent variations in the polypeptide chain of CR1, rather
 than glycosylation state, because they were not abolished by treatment of
 purified receptor protein with endoglycosidase F (Wong, W. W., et al.,
 1983, J. Clin. Invest. 72:685), and they were observed when receptor
 allotypes were biosynthesized in the presence of the glycosylation
 inhibitor tunicamycin (Lublin, D. M., et al., 1986, J. Biol. Chem.
 261:5736). All four CR1 allotypes have C3b-binding activity (Dykman, T.
 R., et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1698; Wong, W. W., et
 al., 1983, J. Clin. Invest. 72:685; Dykman, T. R., et al., 1984, J. Exp.
 Ned., 159:691; Dykman, T. R., et al., 1985, J. Immunol. 134:1787). There
 are four LHRs in the F (or A) allotype of .about.250 kD, termed LHR-A, -B,
 -C, and -D, respectively, 5' to 3' (Wong, et al., 1989, J. Exp. Med.
 169:847). While the first two SCRs in LHR-A determine its ability to bind
 C4b, the corresponding units in LHR-B and -C determine their higher
 affinities for C3b. The larger S (or B) allotype of .about.290 kd has a
 fifth LHR that is a chimera of the 5' half of LHR-B and the 3' half of
 LHR-A and is predicted to contain a third C3b binding site (Wong, et al.,
 1989, J. Exp. Med. 169:847). The smallest F' (or C) allotype of CR1 of
 .about.210 kD, found in increased incidence in patients with systemic
 lupus erthematosis (SLE) and associated with patients in multiple lupus
 families (Dykman, et al., 1984, J. Exp. Med. 159:691; Van Dyne, et al.,
 1987, Clin. Exp. Immunol. 68:570), may have resulted from the deletion of
 one LHR and may be impaired in its capacity to bind efficiently to immune
 complexes coated with complement fragments.
 A naturally occurring soluble form of CR1 has been identified in the plasma
 of normal individuals and certain individuals with SLE (Yoon, et al., 1985
 J. Immunol. 134:3332-3338). Its structural and functional characteristics
 are similar to those of erythrocyte (cell surface) CR1, both structurally
 and functionally. Hourcade, et al. (1988, J. Exp. Med. 168:1255-1270) also
 observed an alternative polyadenylation site in the human CR1
 transcriptional unit that was predicted to produce a secreted form of CR1
 containing the C4b binding domain.
 Several soluble fragments of CR1 have also been generated via recombinant
 DNA procedures by eliminating the transmembrane region from the DNAs being
 expressed (Fearon, et al., International Patent Publication No.
 WO89/09220, Oct. 5, 1989; Fearon, et al., International Patent Publication
 No. WO91/05047, Apr. 18, 1991). The soluble CR1 fragments were
 functionally active, bound C3b and/or C4b and demonstrated factor I
 cofactor activity, depending upon the regions they contained. Such
 constructs inhibited in vitro the consequences of complement activation
 such as neutrophil oxidative burst, complement mediated hemolysis, and C3a
 and C5a production. A soluble construct sCR1/pBSCR1c, also demonstrated in
 vivo activity in a reversed passive Arthus reaction (Fearon, et al., 1989,
 supra; Fearon, et al., 1991, supra; Yeh, et al., 1991 supra), suppressed
 post ischemic myocardial inflammation and necrosis (Fearon, et al.,
 .sup.1989, supra; Fearon, et al., 1991, supra; Weismann, et al., 1990,
 Science, 249:146-151) and extended survival rates following
 transplantation (Pruitt and Bollinger, 1991, J. Surg. Res. 50:350; Pruitt,
 et al., 1991, Transplantation 52:868). [Mulligan et al, 1992, J. Immunol.
 148:3086-3092 (injury following immune complex deposition). Mulligan, et
 al., 1992, J. Immunol. 148:1479-1485 (protection from neutrophil mediated
 tissue injury). Lindsay, et al., 1992, Annals of Surg. 216:677. , Hill, et
 al., 1992, J. Immunol. 149:1722-1728 (tissue ischemia reperfusion
 injuries)].
 CR2. Complement receptor type 2 (CR2, CD21) is a transmembrane
 phosphoprotein consisting of an extracellular domain which is comprised of
 15 or 16 SCRs, a 24 amino acid transmembrane region, and a 34 amino acid
 cytoplasmic domain (Moore, et al., 1987, Proc. Natl. Acad. Sci. U.S.A.
 84:9194-9198; Weis, et al., 1988, J. Exp. Med. 167:1047-1066). Electron
 microscopic studies of soluble recombinant CR2 have shown that, like CR1,
 it is an extended, highly flexible molecule with an estimated contour
 length of 39.6 nanometers by 3.2 nanometers, in which each SCR appears as
 a ringlet 2.4 nanometers in length (Moore, et al., 1989, J. Biol. Chem.
 34:20576-20582).
 By means of recombinant DNA experiments with eukaryotic expression vectors
 expressing deletion or substitution mutants of CR2 in COS cells, the
 ligand 30 binding sites of CR2 have been localized to the two N-terminal
 SCR's of the molecule (Lowell, et al., 1989, J. Exp. Med. 170:1931-1946).
 Binding by cell surface CR2 of the multivalent forms of C3 ligands such as
 iC3b and C3dg causes activation of B-cells (Melchers, et al., 1985,
 Nature, 317:264-267; Bohnsack, et al., 1988, J. Immunol. 141:457-463;
 Carter, et al., 1988, J. Immunol. 143:1755-1760).
 A form of recombinant soluble CR2 has been produced (Moore, et al., 1989,
 J. Biol. Chem. 264:20576-20582). In analogy to the soluble CR1 system,
 soluble CR2 was produced in a recombinant system from an expression vector
 containing the entire extracellular domain of the receptor, but without
 the transmembrane and cytoplasmic domains. This recombinant CR2 is
 reported to bind to C3dg in a 1:1 complex with Kd equal to 27.5 mM and to
 bind to the Epstein-Barr proteins gp350/220 in a 1:1 complex with a Kd of
 3.2 nM (Moore, et al., 1989, J. Biol. Chem. 264:20576-20582).
 CR3. A third complement receptor, CR3, also binds iC3b. Binding of iC3b to
 CR3 promotes the adherence of neutrophils to complement-activating
 endothelial cells during inflammation (Marks, et al., 1989, Nature
 339:314). CR3 is also involved in phagocytosis, where particles coated
 with iC3b are engulfed by neutrophils or by macrophages (Wright, et al.,
 1982, J. Exp. Med. 156:1149; Wright, et al., 1983, J. Exp. Med. 158:1338).
 CR4. CR4(CD11) also appears to be involved in leukocyte adhesion
 (Kishimoto, et al., 1989, Adv. Immunol. 46:149-82).
 DAF. DAF, or decay-accelerating factor, is a membrane protein that appears
 to have similar action to C4Bp in bringing about a functional-dissociation
 of C2b from C4b. DAF is linked to membranes via a phosphatidyl inositol
 glycolipid, and its absence from red blood cells has been shown to be a
 major causative factor in paroxysmal nocturnal hemoglobinuria.
 (Encyclopedia of Human Biology, Academic Press, Inc. 1991). DAF binds to
 C3b/C4b as well as C3 convertases (EP 0512 733 A2).
 DAF contains 4 SCRs followed by an O-linked glycosylation region, and is
 terminated with a glycolipid anchor (EP 0512 733 A2). Cells that express
 DAF show substantial increases in resistance to complement-mediated cell
 lysis (Lublin, D. M. et al., 1991, J. Exp. Med. 174:35; Oglesby, T. J., et
 al., 1991; Trans. Assoc. Am. Phys. CIV:164-172; White, D. J. G., et al.,
 1992; Transplant Proc. 24:474-476).
 MCP. MCP or membrane cofactor protein, like DAF, contains 4 SCRs followed
 by an O-linked glycosylation region. MCP is terminated witn an extra
 cytoplasmic segment (whose importance is unknown) a transmembrane region
 and an intracellular domain (EP 0512 733 A2). Also, like DAF, cells
 expressing NCP confer substantial increases in resistance to
 complement-mediated cell lysis. (EP 0512 733 A2 and Lublin, D. M., et al.,
 J. Exp Med (19) 174:35; Oglesby, T. J. et al., Trans Assoc Am Phys (1991)
 CIV:164-172; White, D. J. G., et al., Transplant Proc (1992) 24:474-476).
 FACTOR H. Factor H is a plasma protein that is exclusively or predominantly
 composed of SCRs (Chung, L. P., et al., 1985, Biochem. J. 230:133;
 Kristensen, T., et al., 1986, J. Immunol. 136:3407). Factor H is a
 regulator of the alternative pathway. Factor H binds to C3b and to the C3b
 portion of C3 convertases (C3b, Bb) (Encyclopedia of Human Biology, supra)
 accelerating dissociation of Bb from these complexes thereby inactivating
 them. Factor H also regulates the use of C5 in the classical pathway by
 competing with C5 for binding to C3b, thus inactivating the activity of
 the C3/C5 convertase (Encyclopedia of Human Biology, supra).
 2.3. SELECTINS AND SELECTIN LIGANDS
 Selectins are a group of cell surface glycoproteins which
 characteristically display a NH.sub.2 terminal lectin domain related to
 the carbohydrate recognition structure described for animal lectins, an
 epidermal growth factor domain, and a domain consisting of short repeating
 sequences analogous to those found in the complement regulatory proteins
 which map to a region of chromosome 1 called the regulators of complement
 activity (RCA) (Harlan & Liu, Adhesion: Its Role in Inflammatory Disease,
 W. H. Freeman & Co., 1992). Three independently studied selecting have
 been characterized and are named according to the cell type upon which
 each was originally identified. Under the current nomenclature there are
 the E-selectins, originally identified on cytokine-activated endothelial
 cells (Bevilacque, M. P. et al., (1985) J. Clin. Invest. 76:2003-2011);
 P-selectins, discovered on activated platelets (Hsu-Lin, P. E., et al.
 (1984) J. Biol. Chem. 259:9121-9126); and finally, L-selectins recognized
 as a cell surface marker on most leukocytes including lymphocytes,
 neutrophils, and monocytes (Kansas, G. S. et al., (1985) J. Immunol.
 134:2995-3002). Each selectin has been implicated as a key factor in
 important events in cellular adhesion and recognition. As such, their
 carbohydrate recognition structures at the NH.sub.2 -terminal portion of
 the molecule as well as their carbohydrate ligands have been extensively
 studied.
 Selectins, then, are cell adhesion molecules that in inflammatory
 situations are responsible for the attachment of platelets and leukocytes
 to vascular surfaces and their subsequent infiltration into the tissue.
 During a normal inflammatory response the leukocytes, in responding to
 various signals, enter the tissue and phagocytize invading organisms. In
 various pathologic inflammatory diseases, such as psoriasis and rheumatoid
 arthritis, this response may lead to serious organ tissue damage.
 Similarly, in reperfusion injury, invading leukocytes are responsible for
 tissue damage. And, aside from their involvement in inflammation, cell
 adhesion molecules on selecting play a central role in other diseases such
 as tumor metastasis.
 In inflammatory situations, all three selecting are implicated in the
 recruitment of leukocytes to the site of inflammation. Early events in the
 inflammatory response include the recruitment of neutrophils to the site
 of tissue damage. In normal situations, circulating lymphocytes bind to
 the vascular endothelium with low avidity. Under situations of distress
 however, as when the body has been invaded by a bacterial pathogen or when
 tissue damage has occurred, leukocytes interact with the activated
 endothelium in another manner. First, up regulation of selecting on
 endothelial cells and platelets occurs to control the localization of
 leukocytes to the inflamed endothelium. The initial step of attachment of
 neutrophils to the endothelial cells lining the venules is controlled by
 selecting and is known as neutrophil "rolling" (von Andrian, U. H. et al.,
 (1991) Proc. Natl. Acad. Sci., U.S.A. 88:7538-7542; Smith, C. W., et al.,
 (1991) J. Clin. Invest., 87:609-618). This "rolling" precedes the firm
 adhesion of leukocytes, especially neutrophils to the endothelium which is
 controlled by a different class of receptors known as the integrins.
 (Lawrence, M. B. and Springer, T. S. (1991) Cell 65: 859-873; von Andrain,
 U. H. et al., (1991) Proc. Natl. Sci. U.S.A. 88:7538-7542; Larson R. S.
 and Springer, T. A. (1990) Immunol. Rev. 114:181-217). Extravasation of
 the cells into the surrounding tissue proceeds after the aforementioned
 attachment processes have each been accomplished.
 One of the selecting, E-selectin (ELAM-1, endothelial cell adhesion
 molecule, LECCAM-2) is expressed on endothelial cells following induction
 by cytokines such as interleukin-1.beta., tumor necrosis factor-.alpha.,
 lymphotoxin, bacterial endotoxins, interferon-.gamma. and the neuropeptide
 substance-p (Harlan & Liu, supra). The expression of E-selectin on
 activated endothelium requires de novo synthesis, peaks at 4-6 hours, and
 persists from 2-48 hours after initial stimulus. Activated endothelia
 expressing the ELAM-1 receptor have been shown to bind neutrophils
 (Bevilacque M. P., et al. (1987) Proc. Natl. Acad. Sci. U.S.A.
 84:9238-9242); monocytes (Walz, G. et al., (1990) Science 250: 1132-1135):
 eosinophil (Kyan-Aung (1991) J. Immunol. 146:521-528): and NK cells
 (Goelz, S. E. (1990) Cell 63:1149-1356). Additionally, activated
 endothelium binds some carcinoma cells (Rice, G. E. and Bevilacqua M. P.
 (1989) Science 246:1303-1306; Walz, G. et al., (1990) 250 1132-1135)
 implicating a role for E-selectins in attachment of tumor cells to blood
 vessel walls.
 P-selectin (CD62, granule membrane protein-140, GMP-140, platelet
 activation dependent granule external membrane, Padgem, LECCAM-3) is
 expressed on activated platelets as well as endothelial cells. The
 P-selectin expression can be mobilized from intracellular stores in
 minutes after activation. P-selectins bind neutrophils and monocytes, as
 well as carcinoma cells (Walz, G., et al., (1990) 250:1132-1135).
 P-selectin, or CD62 expression does not require de novo synthesis because
 this selectin is stored in secretory granules, also called Weibel-Palade
 bodies, in both platelets and endothelial cells. Thus, within minutes of
 activation of either cell type, for example by thrombin, histamine, or
 phorbol esters, CD62 is rapidly transported to the surface of the cell
 where it can bind the ligand found on neutrophils, monocytes, and other
 cells, These ligand-bearing cells then adhere to the platelet or
 endothelial cells expressing the CD62 receptor.
 Patel et al. have found that endothelial cells also express CD62 in
 response to low levels of hydrogen peroxide or other oxidizing agents
 through the production of free radicals (Patel et al., 1991, J. Cell Biol.
 112:749-759). While endothelial cells normally reinternalize CD62 within
 minutes of activation, induction by free radicals produces prolonged
 expression of the selectin. Because neutrophils release oxidizing agents
 and free radicals following activation, initial recruitment of neutrophils
 by transiently expressed CD62 could effectively prolong the expression of
 CD62 through free radical generation by neutrophils (Harlan & Liu,
 Adhesion, supra).
 L-selectin, (lymphocyte homing receptor, LECCAM-1, Mel-14, Leu-8, TQ-1,
 Ly-22, LAM-1) is constitute expressed on the cell surface and is shed
 after activation (Jung. T. M. et al., (1988) J. Immunol., 141:4110-4117).
 Recent advancements in the field of adhesion molecules have led to the
 understanding of the role of protein-carbohydrate interactions. In
 particular, the ligands for selectins have been recently studied
 (Bevilaque, M. P. and Nelson, R. M. (1993) J. Clin. Invest. 91: 379-387).
 Among the ligands identified are the Lewis X blood antigen (Le.sup.x) and
 sialylated Lewis X antigen. The Lewis X antigens have been known for some
 time, and had been identified as the terminal structures on cell surface
 glycoproteins and glycolipids or neutrophils and promyelocytic cell lines
 (Harlan & Liu, Adhesion, supra).
 Lowe et al. demonstrated that transfection of a cDNA for the Lewis blood
 group fucosyl transferase (Gal.beta.1,3/4GlcNAca1,3 fucosyltransferase)
 into Chinese hamster ovary (CHO) cells resulted in the expression of the
 Le.sup.x and SLe.sup.x antigens and the simultaneous ability of the
 transfected cells to adhere to E-selectins on TNF-.alpha.-activated human
 umbilical vein endothelial cells (HUVECs) (Lowe et al., 1990, Cell
 63:475-484). Sialidase treatment of the cells abolished their ability to
 adhere to activated HUVECs, indicating that a sialylated structure was
 required for adhesion. Additionally, it was observed that a pre-myelocytic
 leukemia-60 (HL-60) cell clone which expressed SLe.sup.x bound to HUVECS
 while another clone that did not express SLe.sup.x did not bind to HUVECS.
 Phillips et al. produced CHO glycosylation mutants, which, unlike the
 wild-type cells, expressed fucosyltransferase activities that synthesized
 both Le.sup.x and SLe.sup.x (LEC11) or Le.sup.x only (LEC12) as terminal
 sugar structures on cell surface glycoproteins (Phillips et al., 1990
 Science 250:1130-1132). Only LEC11 cells bound to E-selectin on activated
 HUVECs, and the adhesion was abolished by pretreatment of the LEC11 cells
 with sialidase, implicating SLe.sup.x as the ligand.
 The nucleic acid sequence of an .alpha.1,3-fucosyl transferase responsible
 for adding a fucosyl residue to an appropriate carbohydrate such as ELAM,
 through an .alpha.1,3 glycosidic linkage has been reported (International
 Patent Publication No. WO91/16900). This report also describes recombinant
 COS and CHO cells transformed with the transferase.
 Other ligands that bind to selectins have also been disclosed. These
 ligands structurally resemble the Lewis X antigens (International Patent
 Publication No. WO92/02527 and International Patent Publication No.
 WO91/19502).
 2.4. DISEASES INVOLVING INAPPROPRIATE COMPLEMENT ACTIVITY
 Diminished expression of CR1 on erythrocytes of patients with systemic
 lupus erythematosus (SLE) has been reported by investigators from several
 geographic regions, including Japan (Miyakawa, et al., 1981, Lancet
 2:493-497; Minota, et al., 1984, Arthr. Rheum. 27:1329-1335), the United
 States (Iida, et al., 1982, J. Exp. Med. 155:1427-1438; Wilson, et al.,
 1982, N. Engl. J. Med. 307:981-986) and Europe (Walport, et al., 1985,
 Clin. Exp. Immunol. 59:547; Jouvin, et al., 1986, Complement 3:88-96;
 Holme, et al., 1986, Clin. Exp. Immunol. 63:41-48). CR1 number has also
 been found to correlate inversely with serum levels of immune complexes,
 with serum levels of C3d, and with the amounts of erythrocyte-bound C3dg,
 perhaps reflecting uptake of complement-activating immune complexes and
 deposition on the erythrocyte as an "innocent bystander" (Ross, et al.,
 1985, J. Immunol. 135:2005-2014; Holme, et al., 1986, Clin. Exp. Immunol.
 63:41-48; Walport, et al., 1985, Clin. Exp. Immunol. 59:547).
 Abnormalities of complement receptor expression in SLE are not limited to
 erythrocyte CR1. Relative deficiencies of total cellular CR1 of
 neutrophils and plasma membrane CR1 of B lymphocytes of the SLE patients
 have been shown to occur (Wilson, et al., 1986, Arthr. Rheum. 29:739747).
 The relative loss of CR1 from erythrocytes has also been observed in
 patients with Human Immunodeficiency Virus (HIV) infections (Tausk, F. A.,
 et al., 1986, J. Clin. Invest. 78:977-982) and with lepromatous leprosy
 (Tausk, F. A., et al., 1985, J. Invest. Dermat. 85:58s-61s).
 Complement activation has also been associated with disease states
 involving inflammation. The intestinal inflammation of Crohn's disease is
 characterized by the lymphoid infiltration of mononuclear and
 polymorphonuclear leukocytes. It was found recently (Ahrenstedt, et al.,
 1990, New Engl. J. Med. 322:1345-9) that the complement C4 concentration
 in the jejunal fluid of Crohn's disease patients increased compared to
 normal controls. Other disease states implicating the complement system in
 inflammation include thermal injury (burns, frostbite) (Gelfand, et al.,
 1982, J. Clin. Invest. 70:1170; Demling, et al., 1989, Surgery 106:52-9),
 hemodialysis (Deppisch, et al., 1990, Kidney Inst. 37:696-706; Kojima, et
 al., 1989, Nippon Jenzo Gakkai Shi 31:91-7), and post pump syndrome in
 cardiopulmonary bypass (Chenoweth, et al., 1981, Complement Inflamm.
 3:152-165; Chenoweth, et al., 1986, Complement 3:152-165; Salama, et al.,
 1988, N. Engl. J. Med. 318:408-14). Both complement and leukocytes are
 reported to be implicated in the pathogenesis of adult respiratory
 distress syndrome (Zilow, et al., 1990, clin Exp. Immunol. 79:151-57;
 Langlois, et al., 1989, Heart Lung 18:71-84). Activation of the complement
 system is suggested to be involved in the development of fatal
 complication in sepsis (Hack, et al., 1989, Am. J. Med. 86:20-26) and
 causes tissue injury in animal models of autoimmune diseases such as
 immune complex-induced vasculitis (Cochrane, 1984, Springer Seminar
 Immunopathol. 7:263), glomerulonephritis (Couser et al, 1985, Kidney Inst.
 29:879), hemolytic anemia (Schreiber and Frank, 1972, J. Clin. Invest.
 51:575), myasthenia gravis (Lennon, et al., 1978, J. Exp. Med. 147:973;
 Biesecker and Gomez, 1989, J. Immunol. 142:2654), type II collagen-induced
 arthritis (Watson and Townes, 1985, J. Exp. Med. 162:1878), and
 experimental allergic and hyperacute xenograft rejection (Knechtle, et
 al., 1985, Heart Transplant 4(5):541; Guttman, 1974, Transplantation
 17:383; Adachi, et al., 1987, Trans. Proc. 19(1):1145). Complement
 activation during immunotherapy with recombinant IL-2 appears to cause the
 severe toxicity and side effects observed from IL-2 treatment (This, et
 al., 1990, J. Immunol. 144:2419).
 Complement may also play a role in diseases involving immune complexes.
 Immune complexes are found in many pathological states including but not
 limited to autoimmune diseases such as rheumatoid arthritis or SLE,
 hematologic malignancies such as AIDS (Taylor, et al., 1983, Arthritis
 Rheum. 26:736-44; Inada, et al., 1986, AIDS Research 2:235-247) and
 disorders involving autoantibodies and/or complement activation (Ross, et
 al., 1985, J. Immunol. 135:2005-14).
 Soluble CR1 has been successfully used to inhibit complement activation In
 a number of animal models: Moat, B. P., et al., 1992, Amer. Review of
 Respiratory disease 145:A845; Mulligan, M. S., et al., 1992, J. Immunol.
 148:1479-1485; Yeh, C. G. et. al., 1991, J. Immunol. 146 250-256; Weisman,
 et al., 1990, Science 249:146-51; Pruitt, et al., 1991, Transplantation
 52(5):868-73; Pruitt and Bollinger, 1991, J. Surg. Res. 50:350-55;
 Rabinovici, et al., 1992, J. Immunol. 149:1744-50; Mulligan, et al., 1992,
 J. Immunol. 148:1479-1485; Lindsay, et al., 1992, Annals of Surg. 216:677.
 Studies of Weisman et al (1990, Science 249:146-151) have demonstrated that
 sCR1 can prevent 90% of the generation of C3a and C5a in human serum
 activated by the yeast cell wall component zymosan. Weisman, et al. (1990,
 supra) also utilized sCRI in the rat to inhibit complement activation and
 reduce the damage due to myocardial infarction. Soluble CR1 also appears
 to inhibit the complement dependent process of the reverse Arthus reaction
 (Yeh, et al., 1991, J. Immuno. 146:250-256), and hyperacute xenograft
 rejection (Pruitt, et al., 1991, Transplantation 52:868-873). Recent data
 (Moat, et al., 1992, Amer. Rev. Respiratory Disease 145:A845) indicate
 that sCR1 is of value in preventing complement activation in an
 experimental model of cardiopulmonary bypass in the pig, a situation where
 complement activation has been demonstrated.
 Citation or identification of any reference of Section 2 of this
 application shall not be constructed as an admission that such reference
 is available as prior art to the present invention.
 3. SUMMARY OF THE INVENTION
 According to the present invention, compositions are provided which
 comprise, in their broadest aspect, a complement moiety and a carbohydrate
 moiety. These compositions are useful in treating diseases or disorders
 involving complement, as well as inhibiting a primary event in the
 inflammatory response such as blocking interactions between intercellular
 adhesion molecules and their ligands. In preferred aspects, it is an
 advantage of the present invention that the compositions comprise a ligand
 for intercellular adhesion molecules. The complement moiety can be any one
 of a number of proteins which can bind to a complement component, or which
 are related to a complement receptor type 1 by virtue of containing an SCR
 motif. The carbohydrate moiety can be any one of a number of carbohydrates
 that bind to or prevent interaction with an intercellular adhesion
 molecule. This construct facilitates localization of the complement
 protein to the site of injury, and advantageously allows for, inter alia,
 lower dosage treatment. It is a further advantage of the present invention
 that the same composition can interrupt an initial event in the
 inflammatory response. Therefore, the complement protein comprising a
 cellular adhesion molecule ligand is also useful in treating inflammation
 mediated by intercellular adhesion, as well as complement related diseases
 or disorders.
 The carbohydrate moiety of the compositions of the invention is attached to
 the complement moiety by means of an extracellular event such as a
 chemical or enzymatic attachment, or can be the result of an intracellular
 processing event achieved by the expression of appropriate enzymes. In
 certain embodiments, the carbohydrate moiety will specifically bind to
 intercellular adhesion molecules. In one embodiment, the carbohydrate
 binds to a particular class of adhesion molecules known as the selectins.
 Thus, in a preferred aspect, the invention provides for a composition
 comprising at least one complement moiety and at least one carbohydrate
 moiety, which composition preferentially binds to a particular selectin.
 Among the selecting are E-selectin, L-selectin or P-selectin. Particularly
 preferred embodiments comprise at least one complement moiety and at least
 one carbohydrate moiety wherein said carbohydrate moiety comprises an
 N-linked carbohydrate, preferably of the complex type, and more preferably
 fucosylated and sialylated. In the most preferred embodiments, the
 carbohydrate is related to the Lewis X antigen, and especially the
 sialylated Lewis X antigen.
 In one embodiment, the complement moiety is a protein that contains at
 least one short consensus repeat and more preferably binds a component of
 the complement cascade and/or inhibits an activity associated with
 complement. In a more preferred embodiment, the complement moiety
 comprises all or a portion of complement receptor type 1. Preferably the
 complement protein is soluble complement protein. In a most preferred
 embodiment, the complement moiety is soluble complement receptor type 1
 (sCR1), or a fragment or derivative thereof.
 The present invention further provides pharmaceutical compositions
 comprising at least one complement protein and at least one carbohydrate
 moiety in admixture with a suitable pharmaceutical carrier. In a preferred
 embodiment, the complement protein is soluble and particularly sCR1 or
 fragments or derivatives thereof. In these preferred embodiments, the
 carbohydrate is an N-linked carbohydrate, and preferably fucosylated and
 more preferably fucosylated and sialylated. Of these the Lewis X
 (Le.sup.x) antigen or sialyl Lewis X (sLe.sup.x) antigens are particularly
 preferred.
 The present invention also provides methods for producing the compositions
 described herein. In one preferred embodiment, the Invention provides for
 expressing the complement proteins in a cell which glycosylates the
 complement protein with a Le.sup.x antigen, or preferably a SLe.sup.x
 antigen, and recovering the protein. In another embodiment, the invention
 provides for modifying a complement protein by chemically linking the
 carbohydrate moiety to the protein, wherein said carbohydrate moiety is
 preferably a selectin ligand.
 In yet another embodiment, the invention provides for treating a subject
 with a disease involving undesirable or inappropriate complement activity.
 Such treatment comprises administering to a subject in need of treatment,
 a pharmaceutical composition in accordance with the present invention, in
 an amount and for a period of time suitable to regulate said undesirable
 complement activity. Preferably, the carbohydrate moiety in such
 pharmaceutical compositions are selectin ligands such as Le.sup.x, and
 more preferably the ligand is SLe.sup.x. Treatments with the complement
 protein comprising the selectin ligand include, but are not limited to,
 diseases or disorders of inappropriate complement activation, especially
 inflammatory disorders. Such disorders include but are not limited to
 postischemic reperfusion conditions, infectious disease, sepsis, immune
 complex disorders and autoimmune disease.
 The compositions of the invention can be used in homing the complement
 moiety, preferably CR1, and more preferably sCR1, to adhesion molecules
 such as selectins on activated endothelium, allowing for, inter alia, a
 lower dose as compared to the use of sCR1 alone or its present glycoforms.
 The compositions can then persist at the site of inflammation, and thereby
 prevent further activation. Early neutrophil adhesion events which depend
 on selectin/ligand interaction may also be blocked, Additionally, the in
 vivo half life of the sCR1 may be prolonged. In a specific embodiment, a
 CR1 moiety blocks the convertases C3 and C5 in both the classical and
 alternative pathways, and thus prevents the release of C5a. Preventing the
 release of C5a further inhibits, inter alia, neutrophil activation and
 chemoattraction.
 It is yet another advantage that the compositions presented herein may have
 reduced antigenicity. This may be particularly relevant in the context of
 the preferred embodiments as described herein, as the carbohydrates
 relating to Lewis X antigen may be more "natural" in their glycosylation
 patterning as compared to other carbohydrate structures, e.g. those
 obtained from non-human host cells and the like.
 3.1. ABBREVIATIONS
 CR1--Complement receptor one.
 CR2--Complement receptor two.
 CR3--Complement receptor three.
 CR4--Complement receptor four.
 DAF--Decay-accelerating factor.
 ELAM--Endothelial cell adhesion molecule.
 Le.sup.x --Lewis X antigen.
 LHR--Long Homologous Repeat.
 MCP--Membrane cofactor protein.
 sCR1--Soluble complement receptor one.
 SLe.sup.x --Sialyl Lewis X antigen.
 SCR--Short consensus repeat.
 CD15--Lewis X antigen

5. DETAILED DESCRIPTION
 The present invention is directed to compositions comprising at least one
 complement moiety and at least one carbohydrate moiety. The compositions
 of the invention interact on a cellular level with cells expressing
 appropriate receptors. In certain preferred embodiments, the carbohydrate
 moiety of the compositions will bind to a selectin.
 For the sake of clarity, the present invention is described in detail in
 sections relating to the various components of the compositions, methods
 of producing such compositions as well as pharmaceutical preparations
 thereof, functional assays for measurement of activity of the
 compositions, and methods of diagnosis, treatment and prophylaxis using
 the compositions.
 5.1. COMPLEMENT PROTEINS
 "Complement moiety" within the scope of this invention means any protein
 that contains all or a portion of any protein associated with the
 complement cascade, or a protein that contains at least a portion of a
 short consensus repeat. Certain useful complement proteins are described
 in detail in Sections 2.1 and 2.2 of the Background of the Invention, and
 preferably include but are not limited to complete proteins or any
 fragment of: complement receptor type 1 (CR1), which is the receptor for
 complement components C3b and C4b; complement receptor type 2 (CR2), which
 is the receptor for C3d; complement receptor type 3 (CR3), the receptor
 for iC3b; complement receptor type 4 (CR4), which is specific to iC3b;
 complement receptor type 5 (CR5), which is specific for the C3d portion of
 iC3b, C3dg, and C3d; the C5a receptor (C5a-R); and receptors for C3a and
 C4a. In a preferred aspect, the invention is meant to include those
 members of the family of complement regulatory proteins that contain the
 conserved short consensus repeat (SCR) motif. SCR motifs are found in
 complement receptor type 1 and in several other C3/C4-binding proteins,
 most notably CR2, factor H, C4-binding protein (C4-BP), membrane cofactor
 protein (MCP), and decay accelerating factor (DAF). The genes for factor
 H, C4-BP, CR2, and DAF map to a region on chromosome 1 which has been
 designated "regulators of complement activation" (RCA) (Hourcade, D., et
 al., 1989, Advances in Immunol., 45:381-416). Particular analogues of
 these regulators of complement activation are found in Atkinson, et al.,
 EPO Publication No. 0 512 733 A2, published on Nov. 11, 1992. Thus, in a
 preferred embodiment, the complement protein contains at least one SCR and
 is able to bind to a component of complement. Such complement proteins
 will, in one embodiment, bind to C3b or C4b or a fragment of C3 or C4,
 such as those proteins described above.
 CR1 has been extensively studied, and a structural motif of 60-70 amino
 acids, termed the short consensus repeat (SCR) has been discovered. The
 SCR motif is tandemly repeated 30 times in the F-allotype of CR1, and
 additional repeat cycles occur in other allotypes. The consensus sequence
 of the SCR includes 4 cysteines, a glycine and a tryptophan that are
 invariant among all SCRs. Sixteen other positions are conserved, with the
 same amino acid or a conservative replacement being found in over half of
 the other 30 SCRs (Klickstein, et al., 1987, J. Exp. Med. 165:1095-1112;
 Klickstein et al, 1988, J. Exp. Med., 168:1699-1717; Hourcade et al.,
 1988, J. Exp. Med. 168:1255-1270). The dimensions of each SCR are
 estimated to be approximately 2.5-3.0 nm.times.2 nm.times.2 nm.
 Tandem repeats of SCRs (the same invariant residues and similar spacing
 between cysteines) have been identified in 12 additional proteins of the
 complement systems (Ahearn et al., 1989, Adv. Immunol. 46:183-219). These
 proteins share a capacity for interacting with C3, C4, or C5, the set of
 homologous complement proteins that are subunits of the alternative and
 classical C3-C4 convertases and the membrane attack complex, respectively.
 Complement-related proteins containing SCRs may have activating functions
 (Clr, Cls, Factor B and C2), negative regulatory roles (Factor H, C4-BP,
 DAF, MCP, and CR1), serve as cellular receptors capable of eliciting
 functions of phagocytes and lymphocytes (CR1 and CR2) or promote the
 formation of the complement channel-forming membrane attack complex (C6
 and C7). Thus, the SCR is one of the most characteristic structures of the
 complement system. The finding of SCR's in non-complement proteins, such
 as in an interleukin-2 receptor .alpha. chain, .beta.2-glycoprotein 1, and
 factor XIII does not necessarily indicate a complement-related function,
 although this possibility has not been excluded.
 It is within the scope of the invention that the compositions comprise one
 or more of the aforementioned SCRs, in any combination suitable to obtain
 a desired result. As additional criteria, those forms of the complement
 protein or fragments thereof that are readily absorbed by tissues, that
 are protected from rapid metabolism and/or that provide for prolonged half
 life, are preferentially selected in producing the compositions of the
 invention. One skilled in the art may also effect modifications of the
 protein formulation, to effect absorption. These modifications include,
 but are not limited to, use of a prodrug and chemical modification of the
 primary structure (Wearley, L. L., 1991, Crit. Rev. in Ther. Drug Carrier
 Systems, 8(4):333). In minimizing metabolism of the complement protein and
 thereby increasing the effective amount of protein, such modifications
 include but are not limited to chemical modifications and covalent
 attachment to a polymer (Wearley, L. L., 1991, supra).
 The compositions of the present invention may be part of a delivery system
 such as liposomes. Delivery systems involving liposomes are discussed in
 International Patent Publication No. WO 91/02805 and International Patent
 Publication No. WO 91/19501, as well as U.S. Pat. No. 4,880,635 to Janoff
 et al. These publications and patents provide useful descriptions of
 techniques for liposome drug delivery.
 The genes for the complement related proteins are readily available, for
 instance the nucleic acid sequences and/or genes encoding the complement
 proteins of the present invention are known as, for instance; DAF,
 International Patent Publication No. WO89/01041 published Feb. 9, 1989;
 MCP, Lublin M. D., et al., 1988, J. Exp. Med. 168:181-194; and, CR2, Weis,
 J. J., et al., 1988, J. Exp. Med. 168:1047-1066. The CR1 gene and its
 encoded protein are provided for in International Patent Publication No.
 WO89/09220 published Oct. 5, 1989 and entitled "The Human C3b/C4b Receptor
 (CR1)". Once the gene and its encoded protein are available, any number of
 techniques known in the art can be used to modify the gene itself or its
 encoded proteins. The invention is meant to include such complement
 protein-related fragments, derivatives, and analogues. The complement
 protein-related fragments, derivatives, and analogues for use in the
 composition and formulations of the invention can be produced by various
 methods known in the art. The manipulations which result in their
 production can occur at the gene or protein level, or by methods of
 chemical synthesis. For example, a cloned complement gene can be modified
 by any of numerous strategies known in the art (Maniatis, T., 1982,
 Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,
 Cold Spring Harbor, N.Y.). The complement protein gene sequence can be
 cleaved at appropriate sites with restriction endonuclease(s) followed by
 further enzymatic modification if desired, isolated, and ligated In vitro.
 In the production of the gene encoding a derivative, analogue, or peptide
 related to a complement protein, care should be taken to ensure that the
 modified gene remains within the same translational reading frame as the
 native complement protein gene, uninterrupted by translational stop
 signals, in the gene region where the desired complement
 inhibitory-specific activity is encoded.
 Additionally, the complement protein gene can be mutated In vitro or in
 vivo, to create and/or destroy translation, initiation, and/or termination
 sequences, or to create variations in coding regions and/or form new
 restriction endonuclease sites or destroy preexisting ones, to facilitate
 further in vitro modification. Any technique for mutagenesis known in the
 art can be used, including but not limited to, in vitro site-directed
 mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551), use
 of TABX linkers (Pharmacia), and the like methods.
 Manipulations of the complement protein sequence may also be made at the
 protein level. Any of numerous chemical modifications may be carried out
 by known techniques, including but not limited to specific chemical
 cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
 NaBH.sub.4, acetylation, formylation, oxidation, reduction, and the like.
 In a particular embodiment in which the complement protein is CR1, for
 example, specific modifications of the nucleotide sequence of CR1 can be
 made by recombinant DNA procedures that result in sequences encoding a
 protein having multiple LHR-B sequences. See, e.g., International Patent
 Publication No. WO91/05047, published Apr. 18, 1991. Such valency
 modifications alter the extent of C3b binding which may be important for
 disorders associated with such functions, such as immune or inflammatory
 disorders. For example, full-length CR1 or fragments thereof and related
 molecules which exhibit the desired activity can have therapeutic uses in
 the inhibition of complement by their ability to act as a factor I
 cofactor, promoting the irreversible inactivation of complement components
 C3b or C4b (Fearon, D. T., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:5867;
 Iida, K. and Nussenzweig, v., 1981, J Exp. Med. 153:1138), and/or by the
 ability to inhibit the alternative or classical C3 or C5 convertases.
 In another embodiment, specific portions of the sequences of CR1 that
 contain specific, well defined combinations of LHRs or SCRs can also be
 generated. The activities of these compounds can be predicted by choosing
 those portions of the full-length CR1 molecules that contain a specific
 activity. The resulting fragments should, but need not contain, at least
 one of the functions of the parent molecule. Such functions include but
 are not limited to the binding of C3b and/or C4b, in free or in complex
 forms; the promotion of phagocytosis, complement regulation, immune
 stimulation; the ability to act as a factor I cofactor; promoting the
 irreversible inactivation of complement components C3b or C4b, (Fearon, D.
 T., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:5867; Iida, K. and Nussenweig,
 V., 1981, J. Exp. Med. 153:1138); effecting immune complex clearance
 and/or by the ability to inhibit the alternative or classical C3 or C5
 convertases. In a specific embodiment, the CR1 includes LHR's B, C and D
 and does not include LHR A.
 In addition, analogues and peptides related to complement proteins can be
 chemically synthesized. For example, a peptide corresponding to a portion
 of complement protein which mediates the desired activity (e.g., C3b
 and/or C4b binding, immune stimulation, complement regulation, etc.) can
 be synthesized by use of a peptide synthesizer.
 In particular embodiments of the present invention, such complement
 proteins, including derivatives, analogues or fragments thereof, whether
 produced by recombinant DNA techniques or by chemical synthetic methods,
 include but are not limited to those containing, as a primary amino acid
 sequence, all or part of the amino acid sequence of the native complement
 protein including altered sequences in which functionally equivalent amino
 acid residues are substituted for residues within the sequence, resulting
 in a silent change. For example, one or more amino acid residues within
 the sequence can be substituted by another amino acid of a similar
 polarity which acts as a functional equivalent, resulting in a silent
 alteration. Nonconservative substitutions can also result in functionally
 equivalent proteins.
 In one embodiment, substitutes for an amino acid within the complement
 protein sequence may be selected from other members of the class to which
 the amino acid belongs. For example, the nonpolar (hydrophobic) amino
 acids include alanine, leucine, isoleucine, valine, proline,
 phenylalanine, tryptophan and methionine. The polar neutral amino acids
 include glycine, serine, threonine, cysteine, tyrosine, asparagine, and
 glutamine. The positively charged (basic) amino acids include arginine,
 lysine and histidine. The negatively charged (acidic) amino acids include
 aspartic acid and glutamic acid.
 In a particular embodiment, nucleic acid sequences encoding a fusion
 protein, consisting of a molecule comprising a portion of a complement
 protein sequence plus a non-complement protein sequence, can be produced.
 See, e.g., International Patent Publication No. WO91/05047. For example,
 further modifications of complement proteins containing LHRs or SCRs
 include the generation of chimeric molecules containing portions of the
 LHR and/or SCR sequences attached to other molecules whose purpose is to
 affect solubility, pharmacology or clearance of the resultant chimeras.
 Such chimeras can be produced either at the gene level as fusion proteins
 or at the protein level as chemically produced derivatives. Chimeric
 molecules comprising portions of immunoglobulin chains and complement
 protein can contain Fab or (Fab').sub.2 molecules, produced by proteolytic
 cleavage or by the introduction of a stop codon after the hinge region in
 the heavy chain to delete the F.sub.c region of a non-complement
 activating isotype in the immunoglobulin portion of the chimeric protein
 to provide F.sub.c receptor-mediated clearance of the complement
 activating complexes. Other molecules that may be used to form chimeras
 include, but are not limited to, other SCR containing proteins, proteins
 such as serum albumin, heparin, or immunoglobulin, polymers such as
 polyethylene glycol or polyoxyethylated polyols, or proteins modified to
 reduce antigenicity by, for example, derivatizing with polyethylene
 glycol. Suitable molecules are known in the art and are described, for
 example, in U.S. Pat. Nos. 4,745,180, 4,766,106 and 4,847,325 and
 references cited therein. Additional molecules that may be used to form
 derivatives of the biological compounds or fragments thereof include
 protein A or protein G (International Patent Publication No. WO87/05631
 published Sep. 24, 1987 and entitled "Method and means for producing a
 protein having the same IgG specificity as protein G"; Bjorck, et al.,
 1987, Mol. Immunol. 24:1113-1122; Guss, et al., 1986, EMBO J. 5:1567-1575;
 Nygren, et al., 1988, J. Molecular Recognition 1:69-74). Constructs
 comprising a plurality of short consensus repeats having a complement
 binding site, said constructs attached to an immunoglobulin chain or a
 soluble, physiologically compatible macromolecular carrier, are also
 suitable as the complement moiety taught herein. Preparation of these
 constructs is disclosed in International Patent Publication No.
 WO91/16437, herein incorporated by reference.
 Isolation and recovery of encoded proteins may be effected by techniques
 known in the art. The complement proteins may be isolated and purified by
 standard methods including chromatography (e.g., ion exchange, affinity,
 and sizing column chromatography, high performance liquid chromatography),
 centrifugation, differential solubility, or by any other standard
 technique for the purification of proteins. If the complement protein is
 exported by a cell that is producing it, a particularly efficacious method
 for purification of the protein is as follows: the cell culture medium
 containing protein is subject to the sequential steps of a) cationic
 exchange chromatography, b) ammonium sulfate precipitation, c) hydrophobic
 interaction chromatography, d) anionic exchange chromatography, e) further
 cationic exchange chromatography and f) size exclusion chromatography.
 In a more preferred embodiment, the instant invention relates to soluble
 CR1 molecules. As used herein, the term soluble CR1 molecules means
 portions of the CR1 protein, which, upon expression, are not located in
 the cell surface as membrane proteins. As a particular example, CR1
 molecules which substantially lack the transmembrane region are soluble
 CR1 molecules. In a specific emmbodiment of the invention, an expression
 vector can be constructed to encode a CR1 molecule which lacks the
 transmembrane region (e.g., by deletion of the carboxyl-terminal to the
 Aspartate encoded by the most C-terminal SCR), resulting in the production
 of a soluble CR1 fragment. In one embodiment, such a fragment can retain
 the ability to bind C3b and/or C4b, in free or in complex forms. In a
 particular embodiment, such a soluble CR1 protein may no longer exhibit
 factor I cofactor activity.
 Soluble constructs carrying some or all of the binding sites of CR1 are
 also envisioned. Such constructs will in certain preferred embodiments,
 inhibit activation of complement and the complement dependent activation
 of cells. For example, in a specific embodiment, a soluble CR1 molecule
 can be used which retains a desired functional activity, as demonstrated,
 e.g., by the ability to inhibit classical complement-mediated hemolysis,
 classical C5a production, classical C3a production, or neutrophil
 oxidative burst in vitro. In one embodiment, such a fragment can retain
 the ability to bind C3b and/or C4b, in free or in complex form. The sCR1
 molecule so produced can contain the LHR-A, LHR-B, LHR-C, LHR-D, SCR29,
 SCR30, up to and including the first alanine residue of the transmembrane
 region. In a preferred aspect of the invention, the soluble CR1 protein
 has the characteristics of the protein expressed by a Chinese hamster
 ovary cell DUX B11 carrying plasmid pBSCR1/pTCSgpt as deposited with the
 ATCC and assigned accession number CRL 10052.
 In a further specific embodiment, a CR1 molecule can be produced that lacks
 the LHR-A region of the CR1 molecule. To this end, an expression vector
 can be constructed to encode a CR1 molecule which lacks the transmembrane
 region and SCRs 1-7, resulting in the production of a soluble CR1 fragment
 that would be expected to preferentially inhibit the alternative pathway.
 The expression vector so constructed would be expected to contain sites
 for primarily C3b binding. Therefore, such a construct would be expected
 to preferentially inhibit the alternative complement pathway as assessed
 by the in vitro hemolytic assays described herein.
 In yet another embodiment, an expression vector can be constructed to
 contain only SCR's 1-18 of the complement receptor type 1. Such a
 construct would be expected to have the full function associated with
 complement receptor type 1 by virtue of containing sites for binding C3b
 and C4b. Such a product would be expected to inhibit the classical and
 alternative pathways of complement as assessed by the in vitro assays
 described herein. In yet another embodiment the construct may contain only
 SCR's 15-18. Such a construct would be expected to bind C3b primarily, and
 preferentially inhibit the alternative pathway of complement.
 These constructs, as well as the other constructs of the application can
 have advantages due to differences in glycosylation. Such differences are
 expected to affect such parameters as the in vivo half-life of the
 molecule. One skilled in the art will recognize the potential sites for
 N-linked glycosylation will vary with the products of such constructs.
 Differences in glycosylation can be assessed by the functional assays
 described herein for their ability to block the binding of the natural
 ligand for the particular cellular adhesion molecule.
 The complement proteins of the invention can be assayed by techniques known
 in the art in order to demonstrate their complement-related activity. Such
 assays include but are not limited to the following In vitro tests for the
 ability to interact with complement proteins, to inhibit complement
 activity, or to selectively inhibit the generation of complement-derived
 peptides:
 (i) measurement of inhibition of complement-mediated lysis of cells, for
 instance, red blood cells (IH50 assay)(International Patent Publication
 No. WO92/10096)
 (ii) measurement of ability to inhibit formation of complement activation
 products such as, C5a and C5a des Arg and/or measurement of ability to
 inhibit formation of C3a or C3a des Arg, or measurement of ability to
 inhibit formation of C5b-9, or sC5b-9 (International Patent Publication
 No. WO92/10096)
 (iii) measurement of ability to serve as a cofactor for factor I
 degradation of, for instance, C3b or C4b (Makrides et al., (1992)
 267:24754-24761, Weisman, H. F., et al. (1990) Science, 244:146-151).
 (iv) measurement of ability to bind to C3b or other C3 derived proteins, or
 binding of C4b or other C4b derived proteins (Makrides et al, supra,
 Weisman et al, supra)
 (v) measurement of inhibition of alternative pathway mediated hemolysis
 (AH50 assay)(International Patent Publication No. WO92/10096)
 Any complement protein or fragment, derivative or analog thereof, in
 particular a CR1 protein, that has any one of the activities associated
 with complement receptors is within the scope of this invention as the
 complement moiety of the compositions provided herein.
 Activities normally associated with complement receptor type 1 are well
 documented in the art. For example, for soluble CR1 proteins, such
 activities include the abilities in vitro to inhibit neutrophil oxidative
 burst, to inhibit complement-mediated hemolysis, to inhibit C3a and/or C5a
 production, to bind C3b and/or C4b, to exhibit factor I cofactor activity,
 and to inhibit C3 and/or C5 convertase activity. A representative
 disclosure of activities and assays are described inter alia in
 International Patent Publication No. PCT/US89/01358, published Oct. 5,
 1989 as WO89/09220, supra; and entitled Weissman, et al., 1990, Science
 249:146-151; Fearon, D. T. and Wong, W. W., 1989, Ann. Rev. Immunol.
 1:243; Fearon, D. T., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:5867; Iida,
 K. and Nussenzweig, V., 1981, J. Exp. Med. 153:1138; Klickstein et al.,
 1987, J. Exp. Med., 165:1095; Weiss, et al., 1988, J. Exp. Med.,
 167:1047-1066; Moore, et al., 1987, Proc. Natl. Acad. Sci. 84:9194; Moore,
 et al, 1989, J. Biol. Chem. 264:205-76).
 5.2. CARBOHYDRATE STRUCTURES COMPRISING SELECTIN LIGANDS
 The carbohydrate moiety of the compositions of the present invention may be
 selected from a variety of carbohydrate structures. In preferred
 embodiments, this moiety is responsible for binding the complement moiety
 to particular cell adhesion molecules, such as a selectin. Section 2.3 of
 the Background of the Invention details several selectins that the
 carbohydrate moiety suitably binds to. Carbohydrate moieties that bind to
 intercellular adhesion molecules, including selecting, are well known in
 the art. For instance, International Patent Publication No. WO91/19502
 published Dec. 26, 1991 and entitled "Intercellular Adhesion Mediators";
 International Patent Publication No. WO92/02527 published Feb. 20, 1992
 and entitled "New Carbohydrate-Based Anti-Inflammatory Agents";
 International Patent Publication No. WO92/19735 published Nov. 12, 1992
 and entitled "GLYCAM-1(Spg 50), A Selectin Ligand"; International Patent
 Publication No. WO92/01718 published Feb. 6, 1992 and entitled
 "Functionally Active Selectin-derived Peptides and Ligands for GMP-140";
 International Patent Publication No. WO91/19501 published Dec. 26, 1991
 and entitled "Intercellular Adhesion Mediators" all present disclosure of
 carbohydrate molecules useful in the present invention: the published
 patent applications are herein incorporated by reference. The synthesis
 and processing of carbohydrates is also well known in the art (Hubbard, S.
 C. and Ivatt, R. J. (1981) Ann. Rev. Biochem. 50:555-83 and the references
 cited therein; Goochee, C. F., (1991) Biotechnology, 9:1347-1355, and the
 references cited therein; Kobata, A. (1992) Eur. J. Biochem. 209, 483-501,
 and the references cited therein). Accordingly, the carbohydrate moiety of
 the instant invention can efficiently interact with cell adhesion
 molecules.
 Particular ligands for selectins have also been described (Howard, D. R.,
 et al., (1987) J. Biol. Chem. 262:16830-16837, Phillips, M. L., et al.,
 (1990) Science 250:1130-1132, Walz, G. et al., (1990) Science,
 250:1132-1135, Stanley, P., and Atkinson, P., (1986) J. Biol. Chem.
 263:11374-11381; Butcher, E., (1991) Cell, 67:1033-1036). The Lewis X and
 sialyl Lewis X oligosaccharides have been shown to be particularly
 important in selectin binding. Recent studies have further characterized
 the ligand structures for selectins and note that modifications of the
 Lewis X and sialyl Lewis X oligosaccharide may enhance the interactions
 between the oligosaccharides and the selectins (Bevilacqua, M. P. and
 Nelson, R. M. (1993) J. Clin. Invest. 91:379-387, Nelson, R. M., et al.,
 (1993) J. Clin. Invest. 91:1157-1166, Norgard, K. E. et al., (1993) Proc.
 Natl. Acad. Sci., U.S.A. 90:1068-1072; Imai, Y. et al., (1993) Nature
 361:555-557).
 The carbohydrate moiety of the present invention will now be described with
 reference to commonly used nomenclature for the description of
 oligosaccharides. A review of carbohydrate chemistry which uses this
 nomenclature is found in, Hubbard and Ivatt (1981) supra. This
 nomenclature includes, for instance, Man, which represents mannose;
 GlcNAc, which represents 2-N-acetyl glucosamine; Fuc, which represents
 fucose; Gal, which represents galactose; and Glc, which refers to glucose.
 In preferred embodiments, the carbohydrate moiety comprises sialic acid
 residues. Two preferred sialic acid residues are described in shorthand
 notation by "NeuNAc", for 5-N-acetylneuraminic acid, and "NeuNGc" for
 5-glycolyl neuraminic acid. (J. Biol. Chem., 1982, 257:3347; J. Biol.
 Chem., 1982, 257:3352).
 This method of describing carbohydrates, as will be readily understood by
 one skilled in the art, includes notations for the various glycosidic
 bonds relevant to naming carbohydrates. Therefore, in describing a bond
 linking two or more monosaccharides to form an oligosaccharide, a .beta.
 glycosidic bond between the C-1 of galactose and the C-4 of glucose is
 commonly represented by Gal.beta.1-4Glc. The notation .beta. and .alpha.
 are meant to represent the orientation of the bond with respect to the
 glycosidic ring structure. For the D-sugars, for instance, the designation
 .beta. means the hydroxyl attached to the C-1 is above the plane of the
 ring. Conversely, for the D-sugars, the designation a means the hydroxyl
 group attached to the C-1 is below the plane of the ring. The carbohydrate
 moiety will be described with reference to this shorthand notation.
 In its broadest aspects, carbonydrate structures useful in the present
 invention may be selected from a wide range of structures. Preferably, the
 carbohydrate will interact at some level with an adhesion molecule. For
 example, such moiety will bind to, or prevent the binding of a natural
 ligand to a cellular adhesion molecule, or even displace an endogenously
 occurring ligand. As is well understood in the art, interaction between a
 particular ligand and its receptor is generally described by affinity
 constants. "Binding affinity" is generally measured by affinity constants
 for the equilibrium concentrations of associated and dissociated
 configurations of the ligand and its receptor. The present invention
 contemplates such an interaction between a carbohydrate ligand and its
 endothelial cell adhesion molecule receptor. In general, the binding of
 the carbohydrate moiety should occur at an affinity of about k.sub.a
 =10.sup.4 M.sup.-1 or greater to be useful for the present invention, with
 greater than about 10.sup.8 M.sup.-1 being more preferable, and most
 preferably between about 10.sup.8 M.sup.-1 and about 10.sup.4 M.sup.-1.
 In a particular embodiment, the carbohydrate structure of the present
 invention is a ligand for the class of cell adhesion molecules known as
 selecting. Selectins have been shown to bind to a variety of carbohydrate
 structures which can broadly be classified into three groups. The first
 group includes the N-linked and O-linked carbohydrates. N-linked and
 O-linked carbohydrates differ primarily in their core structures. The
 N-linked carbohydrates all contain a common
 Man.alpha.1-6(Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4GlcNAc.beta.-R core
 structure. Of the N-linked carbohydrates, the most important for the
 present invention are the complex N-linked carbohydrates. Such complex
 N-linked carbohydrates will contain several antennary structures. Thus,
 the mono-, bi-, tri-, tetra-, and penta-antennary outer chains are
 important. Such outer-chain structures provide for additional sites for
 the specific sugars and linkages that comprise the carbohydrates of the
 present invention. N-linked glycosylation refers to the attachment of the
 carbohydrate moiety via GlcNAc to an asparagine residue in the peptide
 chain. Therefore, in the core structure described, R represents an
 asparagine residue. The peptide sequences of the complement moiety,
 asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,
 wherein X is any amino acid except proline are possible recognition sites
 for enzymatic attachment of the N-linked carbohydrate moiety of the
 invention. O-linked carbohydrates, by contrast, are characterized by a
 common core structure, which is the GalNAc attached to the hydroxyl group
 of a threonine or serine.
 The N-linked glycans are formed by a series of complex steps occurring
 intracellularly by a series of enzymes with the addition of appropriate
 sugars. Alternatively, the enzymatic synthesis of the core structures can
 be accomplished extracellularly by chemical and enzymatic steps to produce
 the appropriate carbohydrates. These chemical and enzymatic syntheses have
 been described in the art, for instance in International Patent
 Publication No. WO91/19502 and the references described therein, which is
 incorporated herein by reference.
 Specific glycosyltransferases are important for the final outer chain
 structures of the complex carbohydrates. These glycosyltransferases are
 highly specific for the appropriate monosaccharides. Of particular
 importance to the invention are the enzymes involved in sialylation and
 fucosylation of the Gal.beta.1-4GlcNAc group found in the N-linked and
 O-linked oligosaccharides. It will be understood by one skilled in the art
 that terminal glycosylation sequences differ. Among the various structures
 found in the outer chain moieties of the complex oligosaccharide chains
 are the carbohydrates moieties that are known to bind to particular
 selectins.
 Particularly preferred within the context of the present invention are the
 sialylated, fucosylated N-acetylglucosamines which have both a sialic acid
 and a fucose residue in specific position and linkage. Therefore, the
 oligosaccharides related to the Lewis X (Le.sup.x) carbohydrate
 (Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc) are especially useful. Structures of
 the general formula I are particularity relevant:
 ##STR1##
 Especially significant among this group are the sialylated Lewis X
 carbohydrate determinant (sLe.sup.x)
 Neu5Ac.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc and closely related
 structures, including as well, sLe.sup.a, a structural isomer of
 sLe.sup.x, Neu5Ac.alpha.2,3Gal.beta.1,3(Fuc.alpha.1,4)GlcNAc. Therefore,
 in a particularly preferred embodiment the carbohydrate structure is
 represented by the general formula II:
 ##STR2##
 where R represents the remaining carbohydrate structure and SA represents a
 sialic acid. In a preferred embodiment, the sialic acid is
 5-N-acetylneuraminic acid. In another embodiment, the sialic acid is
 5-glycolyl neuraminic acid.
 Additional examples of specific carbohydrate structures useful in the
 compositions of the invention are disclosed in International Patent
 Publication No. WO92/02527 and can be expressed as follows:
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc
 NeuNAc.alpha.2-6 Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc
 NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c
 NeuNAc.alpha.2-6
 Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc
 NeuNAc.alpha.2-6
 Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-6 Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-6Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3gal.beta.1-4(Fuc.
 alpha.1-3)Glc
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3gal.beta.1-4(Fuc.
 alpha.1-3)Glc
 NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-6Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc
 NeuNAc.alpha.2-6Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc
 NeuNAc.alpha.2-3Gal.beta.1-3GlcNAc.beta.1-4Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c
 NeuNAc.alpha.2-6Gal.beta.1-3GlcNAc.beta.1-4Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c
 NeuNAc.alpha.2-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc
 NeuNAc.alpha.2-6Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc
 NeuNAc.alpha.2-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-6Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-3Gal.beta.1-3GlcNAc.beta.1-4Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-6Gal.beta.1-3GlcNAc.beta.1-4Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-6Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)Glc
 NeuNAc.alpha.2-6Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)Glc
 NeuNAc.alpha.2-3Gal.beta.1-3GlcNAc.beta.1-4Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-6Gal.beta.1-3GlcNAc.beta.1-4Gal.beta.1-4(Fuc.alpha.1-3)GlcNA
 c.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-6Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.
 alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNA
 c
 NeuNAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNA
 c
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-3(Fuc.
 alpha.1-4)GlcNAc
 NeuNAc.alpha.2-6Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-3(Fuc.
 alpha.1-4)GlcNAc
 NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNA
 c.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNA
 c.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-3(Fuc.
 alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-6Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-3(Fuc.
 alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4Glc
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-3(Fuc.
 alpha.1-4)Glc
 NeuNAc.alpha.2-6Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-3(Fuc.
 alpha.1-4)Glc
 NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNA
 c.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNA
 c.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-3(Fuc.
 alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 NeuNAc.alpha.2-6Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-3(Fuc.
 alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4(Fuc.alpha.1-3)Glc
 Methods of chemically and enzymatically synthesizing the carbohydrate
 structure are well known in the art and can be found in International
 Patent Publication No. WO91/19502 which is incorporated herein by
 reference. Additionally, these structures may be obtained by the methods
 described infra.
 As will be described in detail in subsequent sections, these structures may
 be provided on the complement moiety by a variety of mechanisms including
 but not limited to the transfection of the particular complement
 expressing cell with appropriate fucosyltransferase enzymes.
 Alternatively, the structures may be chemically synthesized using
 appropriate fucosyltransferases and sialyltransferases and chemically
 linked to the complement moiety. Such transferases are generally available
 as described below.
 As noted earlier, specific modifications of the selectin ligand may enhance
 the interaction between the carbohydrate determinant and particular
 selecting. Nelson et al. studied the binding interaction of a series of
 oligosaccharides based on the SLe.sup.x and SLe.sup.a (sLe.sup.a may be
 especially significant in tumor metastasis due to its significant
 expression on certain cancer cells) structures (Nelson, et al., (1993) J.
 Clin. Invest. 91:1157-1166). Nelson suggests that both the sialic acid and
 the fucose in specific position and linkage enhance E-selectin recognition
 (Nelson, supra). Both the SLe.sup.a and SLe.sup.a contain a terminal
 sialic acid (Neu5Ac) linked in an .alpha.2-3 linkage to the galactose
 (Gal), which is in turn linked to N-acetylglucosamine (GlcNAc). Both
 structures also contain a fucose coupled to the sub terminal GlcNAc. This
 characteristic structure is generally a part of larger glycoproteins.
 Accordingly, in certain preferred embodiments, the carbohydrate is
 modified to contain at least one sialic acid in conformation with at least
 one fucose residue.
 E-selectin may also bind to oligosaccharides related to SLe.sup.x and
 SLe.sup.a which lack the terminal sialic acid but instead have a sulfate
 group (Yuen, C. T. et al., (1992) Biochemistry 31:9126-9131). Modification
 of this primary structure may have selective advantage of homing the
 carbohydrate moiety to a particular selectin. Therefore, within the scope
 of the present invention are the carbohydrates which lack the terminal
 sialic acid but instead have a sulfate group. Additionally, sulphation of
 glycoproteins may enhance ligand binding to L-selectins (Imai, Y, Lasky,
 L. A., and Rosen, S. D. (1993) Science 361:555-557). In this regard, Yuen
 at al., (1992) Biochemistry 31:9126-91341 is instructive and is hereby
 incorporated by reference.
 Selective oxidization of the sialic acid residues, without affecting the
 underlying oligosaccharide, enhances the interaction with L-selectins as
 described by Norgard et al., (1993) Proc. Natl. Acad. Sci. U.S.A
 90:1086-1072, which is incorporated herewith by reference. Other
 modifications of the primary structure which may result in enhanced
 binding or selective binding of the carbohydrate bearing complement
 protein are also within the scope of this invention.
 Carbohydrate moieties within the scope of the present invention may also
 include carbohydrates that by virtue of structural modifications are
 indicated to provide a stabilized carbohydrate moiety having a structure
 more resistant to metabolic degradation than the corresponding naturally
 occurring carbohydrate moiety. Such modified structures may also exhibit a
 high affinity for the particular targeted cell adhesion molecule. Thus,
 the carbohydrate moiety within the scope of the present invention may also
 encompass carbohydrates specifically designed to gain affinity for
 particular intercellular adhesion molecules. Such carbohydrates can be
 structurally modified carbohydrate or a mimetic of a carbohydrate
 structure such that the structural variant or mimetic has about the same
 or better selectin binding activity, immunogenicity, and antigenicity as
 the corresponding naturally occurring carbohydrate structure. Accordingly,
 any modification to a carbohydrate structure that enhances interactions
 dependent on carbohydrate structures for recognition and adhesion are
 within the scope of the present invention. Certain carbohydrate and
 carbohydrate mimetics that are structural and functional variants of the
 naturally occurring carbohydrates are found for instance in International
 Publication No. WO 93/23031, published Nov., 29, 1993, by Toyokuni, et al.
 Those skilled in the art of carbohydrate chemistry and carbohydrate
 mimetics will also recognize those structures which are suitable within
 the context of the present invention based on the teachings herein.
 The second group of carbohydrates that interact with selectins and that are
 included in the present invention, are the phosphorylated mono and
 polysaccharides such as mannose-6-phosphate. This phosphorylated
 monosaccharide, as well as the high molecular weight yeast derived
 phosphomannon (PPME), appear to exclusively bind partners of the
 L-selectins, as P-selectins and E-selectins do not bind these molecules
 (Bevilacqua, M. P. and Nelson, R. N. (1993) J. Clin. Invest. 91:379-387).
 Finally, some sulfated polysaccharides such as heparin bind to selecting
 (Nelson, R. M. et al., (1993) J. Clin. Invest. 91:1157-1166).
 The present invention contemplates at least one discrete carbohydrate unit
 attached to a portion of the complement moiety. One skilled in the art
 will recognize that a complement protein within the scope of the invention
 may contain several sites of N-linked or O-linked glycosylation for the
 attachment of sugar moieties. Therefore, the invention is meant to include
 one or many carbohydrate units attached to any given complement moiety.
 Within a particular carbohydrate side chain of the carbohydrate moiety of
 the compositions, there will often be several sites for the particular
 primary structures to occur. For instance, the N-linked complex
 carbohydrates contain one or more antennary structures that are possible
 locations for attachment of the specific carbohydrate structures of the
 invention to the complement moiety, and therefore, the amount of
 glycosylation of a particular complement moiety may vary greatly in
 accordance with the biological activity one is attempting to achieve with
 the overall composition.
 Differences in glycosylation patterns of the complement moieties are
 advantageous in aiding one to assess a particular composition based on its
 in vivo activity. Accordingly, various factors such as half-life and
 absorption may be assessed, and a particular composition chosen, based on
 these properties. Conditions that affect glycosylation include but are not
 limited to such parameters as media formulation, cell density,
 oxygenation, pH, and the like. Alternatively, one may wish to amplify a
 particular enzyme, such as those specific transferases involved in adding
 the carbohydrate residues in the appropriate position and linkage.
 Several methods known in the art for glycosylation analysis are useful in
 the context of the present invention. Such methods provide information
 regarding the identity and the composition of the oligosaccharide attached
 to the peptide. Methods for carbohydrate analysis useful in the present
 invention include but are not limited to: lectin chromatography;
 HPAEC-PAD, which uses high pH anion exchange chromatography to separate
 oligosaccharides based on charge; NMR,; mass spectrometry; HPLC; GPC;
 monosaccharide compositional analyses; sequential enzymatic digestion.
 Additionally, three main methods can be used to release oligosaccharides
 from glycoproteins. These methods are 1) enzymatic, which is commonly
 performed using peptide-N-glycosidase F/endo-.beta.-galactosidase; 2)
 .beta.-elimination using harsh alkaline environment to release mainly
 O-linked structures; and 3) chemical methods using anhydrous hydrazine to
 release both N-and O-linked oligosaccharides.
 Several methods presented here and known in the art are useful in
 determining the affinity of the molecules for the particular selectin.
 Generally, a number of methods can be used to assay the ability of the
 compositions of the inventions to inhibit intercellular adhesion mediated
 by selecting. The competition assays described in the Example Section, for
 instance, disclose specific methods. For instance, the ability of the
 carbohydrate-bearing complement protein to inhibit adhesion of the natural
 cellular ligands to the cells expressing the particular selectin can be
 used. Typically, the complement protein of the invention is incubated with
 the selectin bearing cells in the presence of the natural ligand-bearing
 cells, wherein the selectin-bearing cells having been immobilized on a
 solid support. Inhibition of the cellular adhesion is then assessed by
 either calculating the amount of the bound complement moiety or assessing
 the displaced cells. In this regard, HL-60 cells and activated human
 platelets and endothelial cells are especially useful.
 In a preferred embodiment, the complement moiety comprises all or a portion
 of the complement receptor type 1, and especially any soluble fragment of
 complement receptor type 1 as described in Section 5.1 infra. In a
 particularly preferred embodiment, the complement moiety comprises sCR1.
 This protein, in its full-length form, has 25 sites for N-linked
 glycosylation. In this embodiment, carbohydrate side chains are provided
 on the sCR1 molecule, which chains comprise one or more carbohydrate
 structures that can bind to or prevent the binding of a particular ligand
 for an endothelial cell receptor. In particular, these carbohydrate
 moieties are ligand.s for selectins. In a particularly preferred
 embodiment, these carbohydrate moieties are the Lewis X oligosaccharides
 sialylated Lewis X oligosaccharides or a combination of both. One skilled
 in the art will understand that the amount of glycosylation may be varied
 from complete saturation of the available glycosylation sites to just a
 few of such sites.
 5.3 PRODUCTION OF COMPLEMENT PROTEINS COMPRISING A SELECTIN LIGAND
 The present invention provides various methods for production of the
 compositions disclosed and claimed herein, methods for preparation of
 complement protein having selectin binding activity, i.e. comprising a
 selectin ligand such as Le.sup.x, or more preferably SLe.sup.x.
 5.3.1. COTRANSFECTION
 As used herein, the term "cotransfection" refers to introduction of a
 nucleic acid encoding at least one complement moiety and at least one
 nucleic acid encoding an enzyme capable of transferring fucose to a
 lactosamine sequence. This results in co-expression of at least one
 complement moiety and the enzyme in the cells. Useful enzymes include the
 .alpha.1,3 fucosyltransferases. These enzymes useful in adding the
 appropriate sugars in the appropriate linkage include, but are not limited
 to .alpha.1,3 fucosyl transferase, .alpha.2,3 sialyl transferase,
 .alpha.2,6 sialyl transferase, .alpha.2,6 sialyl transferase, .beta.1,4
 galactosyl transferase, .beta.1,3 galactosyl transferase, and .beta.1,4
 N-acetyl glucosyl transferase. These may be readily obtained from Genzyme,
 Inc., Cambridge, Mass., Sigma, St. Louis, Mo., the Albert Einstein College
 of Medicine, New York, N.Y., Biogen, Inc., Cambridge, Mass., or the like
 sources. Genes for such transferases are continuously being cloned and
 more are expected to be readily available in the future.
 In a preferred method, a 1,3-fucosyl transferase has been found
 particularly useful for this purpose. The term ".alpha.1,3-fucosyl
 transferase" as used herein refers to any enzyme that is capable of
 forming the Le.sup.x determinant, e.g., capable of transferring fucose to
 the lactosamine sequence. In particular, the .alpha.1,3-fucosyl
 transferase of the invention can demonstrate any one of the known
 substrate specificities (see Harlan and Liu, Adhesion, supra). Preferably,
 the cell is a mammalian cell, such as COS or Chinese hamster ovary (CHO)
 cells.
 Genes which express .alpha.1,3-fucosyl transferase can be obtained from a
 variety of sources (see Kukowska--Latallo et al., 1990, Genes Dev.
 4:1288-1303; International Patent Publication No. WO91/16900; and Paulson
 & Colley, 1989, J. Biol. Chem 264:17615-17618).
 The nucleic acid coding for at least one complement protein and the nucleic
 acid coding for the .alpha.1,3-fucosyl transferase protein can be inserted
 into an appropriate expression vector, or in two vectors. As used herein,
 the term "expression vector" refers to a vector which contains the
 necessary elements for the transcription and translation of the inserted
 protein-coding sequences. The necessary transcriptional and translational
 signals can be supplied by the native genes and/or their flanking regions.
 A variety of host-vector systems may be utilized to express the
 protein-coding sequence, as long as the system provides for glycosylation
 of the complement moiety(ies) using the co-transfected enzyme system.
 Potential host-vector systems include but are not limited to mammalian
 cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.);
 insect cell systems infected with virus (e.g., baculovirus); or
 microorganisms such as yeast containing yeast vectors. The expression
 elements of vectors vary in their strengths and specificities. Depending
 on the host-vector system utilized, any one of a number of suitable
 transcription and translation elements may be used.
 In one embodiment, the expression vector or vectors contains a replication
 origin. In an alternative embodiment, the vector or vectors, which include
 at least one complement moiety and at least one enzyme, are expressed
 chromosomally, after integration of the complement protein and the enzyme
 (e.g. the .alpha.1,3-fucosyl transferase) coding sequence into the
 chromosome by recombination. One skilled in the art will understand that
 it may be desirable to insert multiple genes encoding various transferase
 enzymes or other enzymes to ensure that at least one of the enzymes so
 inserted will be optimal for purposes described herein. Thus, the
 insertion of a multiplicity of genes encoding enzymes demonstrating a
 differential ability to glycosylate may be preferable to insertion of only
 one such gene. Also, one may desire to cotransfect more than one gene
 encoding a complement related protein to vary the constructs of this
 portion of the compositions of the invention.
 Any method known in the art for the insertion of DNA fragments into a
 vector may be used to construct an expression vector or vectors containing
 at least one gene for expression of a complement protein and at least one
 gene for expression of an appropriate enzyme, and appropriate
 transcriptional/translational control signals. These methods may include
 in vitro recombinant DNA and synthetic techniques and in vivo recombinants
 (genetic recombination).
 Expression of additional nucleic acid sequences encoding complement
 proteins or peptide fragments may be regulated by an additional nucleic
 acid sequence so that the complement proteins or peptides and the gene for
 the enzyme is expressed in a host transformed with the recombinant DNA
 molecule. For example, expression of a complement protein and an
 .alpha.1,3-fucosyl transferase may be controlled by any promoter/enhancer
 element known in the art, but these regulatory elements must be functional
 in the host selected for expression. Promoters which may be used to
 control gene expression include, but are not limited to, the SV40 early
 promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the
 promoter contained in the 3' long terminal repeat of Rous sarcoma virus
 (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase
 promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
 78:1441-1445), the regulatory sequences of the metallothionein gene
 (Brinster et al., 1982, Nature 296:39-42); plant expression vectors
 comprising the nopaline synthetase promoter region (Herrera-Estrella et
 al., Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter
 (Gardner, et al., 1981, Nucl. Acids Res. 9:2871), and the promoter of the
 photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella
 et al., 1984, Nature 310:115-120); promoter elements from yeast or other
 fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase)
 promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase
 promoter, and the following animal transcriptional control regions, which
 exhibit tissue specificity and have been utilized in transgenic animals:
 elastase I gene control region which is active in pancreatic acinar cells
 (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring
 Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology
 7:425-515); insulin gene control region which is active in pancreatic beta
 cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control
 region which is active in lymphoid cells (Grosschedl et al., 1984, Cell
 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al.,
 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control
 region which is active in testicular, breast, lymphoid and mast cells
 (Leder et al., 1986, Cell 45:485-495), albumin gene control region which
 is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
 alpha-fetoprotein gene control region which is active in liver (Krumlauf
 et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science
 235:53-58), alpha 1-antitrypsin gene control region which is active in the
 liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene
 control region which is active in myeloid cells (Mogram et al., 1985,
 Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic
 protein gene control region which is active in oligodendrocyte cells in
 the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2
 gene control region which is active in skeletal muscle (Sani, 1985, Nature
 314:283-286), and gonadotropic releasing hormone gene control region which
 is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
 In a preferred embodiment, at least one complement protein with at least
 one gene for a transferase is expressed in mammalian cells and more
 preferably in Chinese hamster ovary (CHO) cells (See, e.g. Stanley et al.,
 1990, J. Biol Chem. 265:1615-1622).
 In a specific embodiment, genomic DNA from cells and plasmid DNA are
 prepared by standard methods (Maniatis) and dissolved in Tris-EDTA (10:1)
 buffer. If polybrene transfection is used, the cellular genomic DNA is
 sheared. The cells can be transfected by either the polybrene or the
 calcium phosphate method (See, e.g. Stanley et al., 1990, supra).
 The cotransfection can also be accomplished using the DEAE-dextran
 procedure (see Lowe et al., 1990, Cell 63:475-484; Davis et al., Basic
 Methods in Molecular Biology, Elsevier Publishing Co., 1986).
 An expression vector or vectors containing at least one complement moiety
 and at least one nucleic acid insert for any appropriate enzyme, can be
 identified by four general approaches: (a) PCR amplification of the
 desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c)
 presence or absence of "marker" gene functions, and (d) expression of
 inserted sequences. In the first approach, the nucleic acids can be
 amplified by PCR with incorporation of radionucleotides or stained with
 ethidium bromide to provide for detection of the amplified product. In the
 second approach, the presence of a foreign gene inserted in an expression
 vector can be detected by nucleic acid hybridization using probes
 comprising sequences that are homologous to an inserted complement protein
 and .alpha.1,3-fucosyl transferase gene. In the third approach, the
 recombinant vector/host system can be identified and selected based upon
 the presence or absence of certain "marker" gene functions
 (e.g.,.beta.-galactosidase activity, thymidine kinase activity, resistance
 to antibiotics, transformation phenotype, occlusion body formation in
 baculovirus, etc.) caused by the insertion of foreign genes in the vector.
 In a specific example, if a complement protein or an .alpha.1,3-fucosyl
 transferase gene are inserted within the marker gene sequence of the
 vector, recombinants containing the inserts can be identified by the
 absence of the marker gene function. In the fourth approach, recombinant
 expression vectors can be identified by assaying for the activity of the
 gene product expressed by the recombinant. Such assays can be based, for
 example, on the physical or functional properties of the gene products in
 vitro assay systems, e.g., complement inhibitory activity, or binding with
 antibody or a selectin (see Section 5.1, supra, and Section 5.3 infra).
 Once a particular recombinant DNA molecule is identified and isolated,
 several methods known in the art may be used to propagate it. Once a
 suitable host system and growth conditions are established, recombinant
 expression vectors can be propagated and prepared in quantity. As
 previously explained, the expression vectors which can be used include,
 but are not limited to, the following vectors or their derivatives: human
 or animal viruses such as vaccinia virus or adenovirus; insect viruses
 such as baculovirus; yeast vectors; and plasmid and cosmid DNA vectors, to
 name but a few.
 In addition, a host cell strain may be chosen which modulates the
 expression of the inserted sequences in addition to adding the
 carbohydrate moiety (e.g. a selectin ligand) or modifies and processes the
 gene product in the specific fashion desired. Expression from certain
 promoters can be elevated in the presence of certain inducers; expression
 of the genetically engineered complement moiety and the enzyme product may
 be controlled. Furthermore, different host cells have characteristic and
 specific mechanisms for the translational and post-translational
 processing and modification (e.g., glycosylation, cleavage [e.g., of
 signal sequence]) of proteins. Appropriate cell lines or host systems can
 be chosen to ensure the desired modification and processing of the foreign
 proteins expressed.
 A vector or vectors containing at least one complement protein and at least
 one nucleic acid sequence encoding an appropriate enzyme are introduced
 into the desired host cells by methods known in the art,. e.g.,
 transfection, electroporation, microinjection, transduction, cell fusion,
 DEAE dextran, calcium phosphate precipitation, lipofection (lysosome
 fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et
 al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem.
 263:14621-14624; Hartmut et al., Canadian Patent Application No.
 2,012,311, filed Mar. 15, 1990).
 Both cDNA and genomic sequences can be cloned and expressed.
 Once a recombinant which expresses the complement protein gene or genes
 with the appropriate enzyme gene or genes is identified, the gene products
 should be analyzed. This can be achieved by assays based on the physical,
 immunological, or functional properties of the product.
 Recovery of the expressed protein product comprising the compositions of
 the invention may be achieved by standard methods of isolation and
 purification, including chromatography (e.g., ion exchange, affinity, and
 sizing column chromatography, high pressure liquid chromatography),
 centrifugation, differential solubility, or by any other standard
 technique for the purification of proteins.
 Any human cell can potentially serve as the nucleic acid source for the
 molecular cloning of the complement moiety gene or genes and the enzyme
 gene or genes. Isolation of the genes involve the isolation of those DNA
 sequences which encode a protein displaying complement protein associated
 structure or properties, e.g., binding of C3b or C4b or immune complexes,
 modulating phagocytosis, immune stimulation or proliferation, and
 regulation of complement. The DNA may be obtained by standard procedures
 known in the art from cloned DNA (e.g., a DNA "library"), by chemical
 synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments
 thereof, purified from the desired human cell (See, for example, Maniatis
 et al., 1982), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
 Laboratory, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA
 Cloning: A Practical Approach, NRL Press, Ltd., Oxford, U.K. Vol. I, II).
 Cells which can serve as sources of nucleic acid for cDNA cloning of the
 genes include but are not limited to monocytes/macrophages, granulocytes,
 B cells, T cells, splenic follicular dendritic cells, and glomerular
 podocytes. Clones derived from genomic DNA may contain regulatory and
 intron DNA regions in addition to coding regions; clones derived from cDNA
 will contain only exon sequences. Whatever the source, the genes should be
 molecularly cloned into a suitable vector for propagation of the gene.
 In a preferred embodiment, the C3b/C4b receptor (CR1) protein and the Lewis
 X antigen are used. In a more preferred embodiment, the CR1 protein and
 the sialyl Lewis X antigen is used. The CR1 gene and its encoded protein
 are provided for in International Patent Publication No. WO89/09220
 published Oct. 5, 1989 and entitled "The Human C3b/C4b Receptor (CR1)". A
 suitable enzyme is .alpha.1,3-fucosyl transferase, whose gene and encoded
 protein are provided for in Lowe et al., 1992, J. Biol. Chem.
 267:4152-4160. Other genes capable of expressing .alpha.1,3-fucosyl
 transferases are described in International Patent Publication No.
 WO91/16900 Kukowska- Latallo et al., 1990, Genes Dev. 4:1288-1303, and
 Paulson et al., 1989, J. Biol. Chem. 264:17615-17618.
 In this preferred embodiment, selection of the cells co-transfected with
 .alpha.1,3-fucosyl transferase that are capable of glycosylating proteins
 with the appropriate carbohydrate molecule can proceed by panning the
 cells with the CD15 structure over platelets activated with thrombin.
 Platelets activated with for instance, thrombin, ADP, collagen, or
 epinephrine, express the selectin receptors CD62/PADGEM/GMP140. Bound
 cells are removed in the presence of a chelating agent such as EDTA since
 the selectin/carbohydrate interaction is dependant on Ca++ and Mg++. These
 released cells are then cloned and screened for the appropriate activity.
 In another embodiment, the cells under consideration can be assayed for
 selectin binding activity in a competitive assay for binding of HL60 or
 U937 cells to activated platelets.
 Assays for directly screening for .alpha.1,3-fucosyl transferase activity
 can be accomplished by a variety of means. For example, an assay can test
 the ability of the .alpha.1,3-fucosyl transferase to link radioactively
 labelled fucose to an acceptor molecule (See International Patent
 Publication No. WO91/16900). Assays which test for .alpha.1,3-fucosyl
 transferase activity are also known in the art (see Stanley et al., J.
 Biol Chem., 1987, 262:16830-16837, Lowe et al., 1992, J. Biol Chem.
 267:4152-4160; Stanley et al., 1990, J. Biol Chem 265:1615-1622).
 5.3.2. MUTAGENESIS
 This invention also encompasses the use of chemical mutagenesis, by well
 known methods in the art, of an appropriate cell line that expresses a
 complement protein to yield a cell line capable of producing a composition
 in accordance with the present invention and preferably a composition,
 comprising a complement protein and selectin ligand, such as the Le.sup.x
 antigen, and more preferably a SLe.sup.x antigen. One suitable method
 envisioned in this invention is production of cell lines that express a
 suitable enzyme, such as .alpha.1,3-fucosyl transferase, using ethyl
 methane sulfonate (Stanley et al., 198:3, Somatic Cell Genetics
 9:593-608).
 Parental cell lines, such as CHO, which express the desired complement
 protein can be mutagenized at 34.degree. C. and 38.5.degree. C. with ethyl
 methane sulfonate (EMS; Eastman Chemical Co., Rochester, N.Y.) at a
 concentration of 100 .mu.g/ml. Any cell line, preferably a mammalian cell
 line, which expresses the desired complement protein can be used, provided
 that mutagenesis can potentially induce one or more enzymes, such as the
 .alpha.1,3-fucosyl transferases, to glycosylate the complement protein.
 For example, cells which endogenously express the complement protein can
 be used, as well as transfected cells which are competent in expressing
 the desired complement protein. See Example Section 6.4.1. for a specific
 embodiment.
 Other methods of mutagenesis are well known in the art and can also be used
 (Maniatis, Ad, supra). Additionally, the cells may be subject to irradium
 and mutagenized cells selected in the techniques described herein.
 Methods for screening for mutagenized cells that express the compositions
 of the invention are known in the art and are described in Section 5.2 and
 the sections following this one.
 5.3.3. TRANSFECTION OF CELLS HAVING APPROPRIATE ENZYME ACTIVITY WITH A
 COMPLEMENT PROTEIN
 Cells expressing transferase enzymatic activity can be obtained from many
 sources. For example, cells can be used which endogenously express an
 appropriate enzyme such as .alpha.1,3-fucosyl transferase, or cells can be
 transfected with genes encoding such enzymes by the methods taught in
 Section 5.3.1 supra. Also, cells which have been previously mutagenized to
 express enzymes necessary for glycosylation with a carbohydrate moiety,
 such as .alpha.1,3-fucosyl transferase can be used (See Section 5.3.2
 supra). or, previously transfected cells can also be used. Once cells
 expressing the appropriate enzyme are obtained they are transfected with
 the complement protein gene by methods known in the art. In particular,
 complement proteins are described in Section 5.1, supra; methods of
 introducing nucleic acids encoding such proteins into a suitable host cell
 that already expresses an appropriate enzyme activity are described in
 Section 5.3.1. , supra.
 In particular, it is envisioned that a nucleic acid encoding a complement
 protein can be introduced into cell lines, which may then express the
 compositions of the invention, and especially the HL-60 (ATCC #CCL-240)
 and K562 (ATCC #CCL-243) cell lines.
 5.3.4. CELL FUSION
 Another method for obtaining the compositions of the invention is by cell
 fusion. The necessary competent cell lines which express an appropriate
 enzyme and a complement protein, i.e., as described in Section 5.3.1.
 through 5.3.3. supra, can be fused with each other using standard cell
 fusion techniques (see Current Protocols in Molecular Biology, Greene and
 Wiley-Interscience (1989)).
 In a specific embodiment, cells that express a complement protein are fused
 with cells that have the enzymatic activity. A specific example of this
 embodiment is presented in the Example Sections below.
 Preferably, when preparing the hybrid cells in accordance with such cell
 fusion techniques, one cell should be selected or engineered, e.g. via
 mutagenesis, to lack the hypoxanthine-guanine phosphoribosyl transferase
 gene. These cells will lack the activity to recycle purine via the salvage
 pathway which utilizes PRPP. These cells should be provided in excess so
 that fusion events will be unlikely to yield hybrid cell-lines which do
 not contain the mutant cells. Either cell line, e.g. the one which
 expresses .alpha.1,3-fucosyl transferase or the cell line which expresses
 complement protein, may be mutagenized. The cells which were not
 mutagenized will maintain the ability to utilize the salvage pathway.
 Therefore, only the few hybrid cell-lines which do not contain the
 mutagenized cell line will survive in the HAT medium (due to the presence
 of the aminopterin). By overwhelming the fusion with the mutagenized
 cells, most of the non-mutagenized cells ("normal" cells) will fuse with
 the mutagens, only a few of the normal cells will not have fused with the
 mutagens. All of the cells which result in a fusion of mutagen: mutagen
 will soon die off since these cells will have no means of utilizing the
 purine salvage pathway. Thus, this negative selection will yield hybrid
 cell-lines which express .alpha.1,3-fucosyl transferase activity and
 complement protein.
 5.3.5. IN VITRO MODIFICATIONS
 The necessary competent cell lines which express an appropriate complement
 protein as described supra, can be used as a source of the complement
 protein for subsequent post-production modification, modification of the
 existing carbohydrate structures may be accomplished using any of the
 appropriate enzymes described supra, at, for instance, Section 5.3.1. In a
 particular embodiment post-production modification occurs in vitro under
 the appropriate conditions using GDP-fucose and the appropriate .alpha.1,3
 fucosyl transferase. Such transferases are described supra. The
 modification described would be expected to yield a fucosylated
 oligosaccharide on an existing core carbohydrate structure such as
 Gal.beta.1-4 GlcNAc. The appropriate sialyl transferase along with the
 appropriate sialic acids would be expected to add the terminal sialic acid
 residues to the appropriate core structures such as Gal.beta.1-4GlcNAc or
 Gal.beta.1-4(Fuc.alpha.1-4) GlcNAc. The resulting carbohydrates can be
 analyzed by any method known in the art including those described herein.
 5.3.6. CHEMICAL MODIFICATION
 The present invention further contemplates preparing the compositions of
 the invention by covalently coupling a carbohydrate moiety to the
 complement moiety using chemical synthesis techniques well known in the
 art.
 Thus, complement protein of this invention can be glycosylated with the
 carbohydrate ligand by chemical modification. This modification can result
 in a glycoprotein in which the complement protein is directly linked to
 the carbohydrate ligand or, in an alternative embodiment, an inert protein
 that has binding activity can be covalently cross-linked to the complement
 protein, whereby the inert protein bridges to the carbohydrate. If such an
 inert protein is used, it is preferably a short consensus repeat (SCR)
 since the SCR is a structural motif found on many complement proteins (see
 Section 5.1. supra), and therefore is likely to minimally affect the
 structure and function of the complement protein.
 As can be appreciated by one of ordinary skill in the art, a carbohydrate
 moiety can be purified and collected from natural sources. An example of
 this process is disclosed for Le.sup.x and sLe.sup.x in Stanley et al., J.
 Biol Chem. 263:11374 (1988), and see WO 91/19502 (PCT/US91/04284) and WO
 92/02527 (PCT/US91/05416). Purified complement protein can also be
 obtained, as described in Section 5.1, supr. Alternatively, the
 carbohydrate moiety can be prepared synthetically (see Wong et al., 1992,
 J. Am. Chem. Soc. 114:9283, C. F. Borman, 1992, C & EN Dec. 7; p25).
 The carbohydrate moiety from any source can be conjugated to the complement
 protein from any source, to obtain the compositions of this invention
 using chemical synthesis techniques. En particularly preferred
 embodiments, the carbohydrate moiety is Lewis X, and more preferably it is
 sialyl Lewis X. Preferably, the complement protein is CR1, and more
 preferably, soluble CR1.
 The chemical cross-linking of the selectin ligand to the complement protein
 can proceed using a traditional cross-linking agent, such as, but not
 limited to molecules having one functional group that can react more than
 one time in succession, such as formaldehyde (although formaldehyde is not
 indicated for use due to its potential carcinogenicity), as well as
 molecules with more than one reactive group. As used herein, the term
 "reactive group" refers to a functional group on the cross-linker that
 reacts with a functional group on the complement protein so as to form a
 covalent bond between the cross-linker and protein. The cross-linker
 should have a second functional group for reacting with the carbohydrate
 moiety. The term "functional group" retains its standard meaning in
 organic chemistry. Preferably the cross-linking agent of the invention is
 a polyfunctional molecule, i.e., it includes more than one reactive group.
 The polyfunctional molecules that can be used are biocompatible linkers,
 i.e., they are non-carcinogenic, nontoxic, and substantially
 non-immunogenic in vivo. Polyfunctional cross-linkers such as those known
 in the art and described herein can be readily tested in animal models to
 determine their biocompatibility.
 The polyfunctional molecule is preferably bifunctional. As used herein, the
 term "bifunctional molecule" refers to a molecule with two reactive
 groups. The bifunctional molecule may be heterobifunctional or
 homobifunctional Preferably, the bifunctional molecule is
 heterobifunctional, allowing for vectorial conjugation of the carbohydrate
 moiety and the complement moiety. Typically, the polyfunctional molecule
 covalently bonds with an amino or a sulfhydryl group on the complement
 protein and a hydroxyl group, an amino an aldehyde or a carboxylic acid on
 the carbohydrate moiety. However, polyfunctional molecules reactive with
 other functional groups on the complement protein, such as carboxylic
 acids or hydroxyl groups, are contemplated in the present invention.
 The homobifunctional molecules have at least two reactive functional
 groups, which are the same. The reactive functional groups on a
 homobifunctional molecule include, for example, aldehyde groups and active
 ester groups. Homobifunctional molecules having aldehyde groups include,
 for example, glutaraldehyde (Poznansky et al., 1984, Science
 223:1304-1306) and subaraldehyde. Homobifunctional molecules having at
 least two active ester units include esters of dicarboxylic acids and
 N-hydroxysuccinimide. Some examples of such N-succinimidyl esters include
 disuccinimidyl suberate and dithio-bis-(succinimidyl propionated), and
 their soluble bis-sulfonic acid and bis-sulfonate salts such as their
 sodium and potassium salts. These chemicals homobifunctional reagents are
 available from Pierce Chemicals, Rockford, Ill.
 When a reactive group of a hetero-bifunctional molecule forms a covalent
 bond with an amino group, the covalent bond will usually be an amido or
 more particularly an imido bond. The reactive group that forms a covalent
 bond with amino groups may, for example, be an activated carboxylate
 group, a halocarbonyl group, or an ester group. The preferred halocarbonyl
 group is a chlorocarbonyl group. The ester groups are preferably reactive
 ester groups such as, for example, an N-hydroxy-succinimide ester group or
 that of N-maleimido-6-aminocaproyl ester of
 1-hydroxy-2-nitrobenzene-4-sulfonic acid sodium salt (Mal-Sac-HNSA; Bachem
 Biosciences, Inc.; Philadelphia, Pa.).
 Another functional group on the complement protein typically is either a
 thiol group, a group capable of being converted into a thiol group, or a
 group that forms a covalent bond with a thiol group. Free sulfhydryl
 groups can be generated from the disulfide bonds of a complement protein
 (or peptide) that contains one or more disulfides. This is accomplished by
 mild reduction of the protein molecule. Mild reduction conditions are
 preferred so that the secondary and tertiary structure of the protein is
 not significantly altered so as to interfere with the protein function.
 Excessive reduction could result in denaturation of the protein. Such
 reactive groups include, but are not limited to, disulfides that can react
 with a free thiol via disulfide transfer, e.g., pyridyl disulfide,
 p-mercuribenzoate groups and groups capable of Michael-type addition
 reactions (including, for example, maleimides and groups of the type
 described in Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101:3097-3110).
 The covalent bond will usually be a thioether bond or a disulfide. The
 reactive group that forms a covalent bond with a thiol group may, for
 example, be a double bond that reacts with thiol groups or an activated
 disulfide. A reactive group containing a double bond capable of reacting
 with a thiol group is the maleimido group, although others, such as
 acrylonitrile, are also possible. A reactive disulfide group may, for
 example, be a 2-pyridyldithio group or a 5,5'-dithio-bis-(2-nitrobenzoic
 acid) group.
 According to the present invention, for attachment to sulfhydryl groups of
 reduced proteins, the substrate linkers can be modified by attaching a
 maleimide or disulfide group to one end of the linker. The unmodified site
 on the linker is covalently attached to a functional group on the
 carbohydrate moiety. For instance, the substrate linkers which are ester
 or amide linked to compounds as described (Partis et al., 1983, J. Pro
 Chem. 2:263; Means and Feeney, 1990 Bioconjugate Chem. 1:2-12).
 Some examples of heterobifunctional reagents containing reactive disulfide
 bonds include N-succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson, et
 al., 1978, Biochem J., 173:723-737), sodium
 S-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and
 4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene. Some
 examples of heterobifunctional reagents comprising reactive groups having
 a double bond that reacts with a thiol group include succinimidyl
 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and succinimidyl
 m-maleimidobenzoate.
 Other heterobifunctional molecules include succinimidyl
 3-(maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate,
 sulfosuccinimidyl 4-(N-maleimidomethyl-cyclohexane)-1-carboxylate, and
 maleimidobenzoyl-N-hydroxy-succinimide ester. Many of the above-mentioned
 heterobifunctional reagents and their sulfonate salts are available from
 Pierce Chemicals, (supra). Additional information regarding how to make
 and use these as well as other polyfuctional reagents that may be obtained
 are well known in the art. For example, methods of cross-linking are
 reviewed by Means and Feeney, 1990, Bioconjugate Chem. 1:2-12.
 The reactive groups of the cross-linking agent can be spaced via an alkyl
 (including saturated and unsaturated) group, a cyclic alkyl group, a
 substituted alkyl or cyclic alkyl group, or an equivalent spacer group,
 including a peptide sequence. In a specific embodiment, the cross-linking
 reactive groups are spaced from 0 to about 20 atoms from each other,
 although spaces of more than 20 atoms are also contemplated.
 In another embodiment, carbohydrate side chains of complement glycoproteins
 may be selectively oxidized to generate aldehydes (see, e.g., Jackson,
 1944, Organic Reactions 2:341; Bunton 1965, Oxidation in organic
 Chemistry, Vol. 1 (Wiberg, ed.), Academic Press, New York, p. 367;
 (Cooper, et al., 1959, J. Biol. Chem. 234:445-448). This is preferred when
 the carbohydrate side chains are not selectin ligands. The resulting
 aldehydes may then be reacted with amine groups (e.g., ammonia derivatives
 such as a primary amine, hydroxylamine, hydrazide, hydrazide,
 thiohydrazide, phenylhydrazine, semicarbazide or thiosemicarbazide) to
 form a Schiff base or reduced Schiff base (e.g., imine, oxime, hydrazone,
 phenylhydrazone, semicarbazone or thiosemicarbazone, or reduced forms
 thereof).
 Hydrazide cross-linking agents can be attached to a selectin ligand, e.g.,
 Le.sup.x or SLe.sup.x, via an ester or amide link or a carbon-carbon bond,
 and then reacted with an oxidized complement glycoprotein, containing an
 oxidized carbohydrate. This results in hydrazone formation and the
 covalent attachment of the compound to the carbohydrate side chain of the
 glycoprotein via a cross-linker group.
 Alternatively, a glycoprotein form of a complement protein can be reacted
 with an .alpha.1,3-fucosyl transferase in the presence of fucose to yield
 a fucosylated form of the complement inhibitory glycoprotein.
 5.4. FUNCTIONAL ACTIVITY
 The present invention further provides assays for evaluating the functional
 activity of the compositions of the invention. In particular, the
 invention provides certain useful functional assays for a CR1 molecule
 comprising a selectin ligand such as Le.sup.x, or preferably SLe.sup.x. As
 used herein, the term "functional activity" refers to immunological
 binding in addition to biological functions of a molecule.
 Physical-chemical assays are also envisioned for determining the nature of
 the complement moiety and the carbohydrate moiety of the compositions of
 the invention.
 In one embodiment, the activities of the complement moiety and the
 carbohydrate moiety can be evaluated separately. Thus, a complement
 protein comprising a carbohydrate moiety in accordance with the teachings
 herein may have similar or identical electrophoretic migration,
 isoelectric focusing behavior, proteolytic digestion maps, C3b and/or C4b
 and/or immune complex binding activity, complement regulatory activity,
 effects on phagocytosis or immune stimulation, or antigenic properties as
 known for the complement inhibitory protein, e.g., as described in Section
 5.1, supra. Similarly, the functional activity of the carbohydrate moiety
 can be assayed directly, e.g., as described in Section 5.2, supra, and in
 International Patent Publication No. WO91/19502.
 A number of currently available monoclonal antibodies can be used according
 to the present invention to inhibit intercellular adhesion mediated by
 selecting. For instance, CSLEX-1 (see, Campbell et al., J. Biol. Chem.
 259:11208-11214 (1984)), VIM-2, which recognizes a sequence slightly
 different from SLe.sup.x (see, Macher et al., supra), FH6 (described in
 U.S. Pat. No. 4,904,596) (all references are incorporated herein by
 reference) or SH.sub.3 and SH.sub.4 generated by Dr. S. Hakomori of the
 Biomembrane Institute in Seattle, Wash.
 In another embodiment, the functional activities or physical-chemical
 properties of the complement moiety and the carbohydrate moiety are
 evaluated in the same assay. For example, in a specific embodiment, the
 molecular weight of a complement inhibitory protein comprising a selectin
 ligand can be estimated by PAGE, an increase in the apparent molecular
 weight indicating attachment of the selectin ligand, such as Le.sup.x, or
 preferably SLe.sup.x, to the protein. In another embodiment, a sandwich
 immunoassay can be used to assay the functional activity. For example, by
 using antibodies to a complement inhibitory protein and a selectin ligand,
 the composition may be identified. In a specific embodiment, infra, an
 antibody specific for CR1 is adsorbed to an assay plate. The putative
 soluble CR1 comprising the selectin ligand SLe.sup.x or Le.sup.x is added
 to the plate under conditions that allow antibody binding. The presence of
 bound soluble CR1 comprising SLe.sup.x or Le.sup.x is detected by adding a
 CSLE.sup.x antibody or anti-CD15 antibody, respectively, labelled with
 FITC, followed by an anti-FITC antibody labelled with horseradish
 peroxidase. As will be readily understood by one of ordinary skill in the
 art, such sandwich immunoassay can be configured with the CLSE.sup.x
 antibody or anti-CD15 antibody on the solid phase, or as a direct rather
 than an indirect assay. In yet a further embodiment, a Western Blot assay
 can be used to show that the product includes a complement protein
 comprising a selectin ligand. In one aspect, the apparent molecular weight
 of a protein detected in one lane with an antibody to the complement
 protein and in another lane with an antibody to the selectin ligand can be
 compared. Results showing identical molecular weight are indicative of a
 positive identification of the molecule. In another aspect, the protein
 can be purified by affinity chromatography, either on an anti-complement
 protein column or an anti-selectin ligand column, and the purified protein
 detected on Western blotting with the alternative protein.
 As can be readily appreciated by one of ordinary skill in the art, any
 affinity binding partner of a complement inhibitory protein or a selectin
 ligand of high enough affinity can be used in assays in place of specific
 antibody molecules.
 One skilled in the art will also understand that there may be other ways
 the activity of the individual components of the compositions may be
 assayed, or the overall activity of the compositions as a whole may be
 assayed. These types of assays are informational with respect to achieving
 the desired overall functions of the compositions in a desired setting,
 such as the therapeutic arena. Accordingly, this Section, as well as the
 Examples Section, is meant to be exemplary of certain well-accepted
 techniques.
 5.5. THERAPEUTIC COMPOSITIONS AND USES
 One major advantage of the compositions of the present invention is that
 the carbohydrate moiety "homes" to inflamed endothelium, and thus
 localizes the composition to the site of tissue damage, thereby
 potentiating its anti-complement activity and also blocking
 neutrophil-endothelial cell interactions such as neutrophil rolling and
 extravasation. By providing for the homing of the complement protein to
 the site of injury, resulting in its persistence there, the claimed
 compositions advantageously allow for lower dosage treatment than would be
 possible when dosing with either of the constituents alone. The
 compositions of the invention may also demonstrate an increased half life
 in vivo and/or a great bioavailability.
 Expression of selecting participates in the recruitment of cells to sites
 of inflammation. It is well-documented that multiple adhesion proteins and
 their ligands are required for the process of leukocyte adhesion to and
 extravasation across endothelial cells. For example, based on studies
 performed with known activators of the expression of ELAM-1 (inflammatory
 cytokines, endotoxin) and CD62 (thrombin, histamine, etc.), their
 expression is thought to represent inflammatory and hemostatic responses
 to tissue injury.
 Leukocyte traffic across the vessel walls to extravascular vascular tissue
 is necessary for host defense against microbial organisms or foreign
 antigens and repair of tissue damage. Under some circumstances, however,
 leukocyte-endothelial interactions may have deleterious consequences for
 the host. During the process of adherence and transendothelial migration,
 leukocytes may release products such as oxidants, proteases, or cytokines
 that directly damage endothelium or cause endothelial damage by releasing
 a variety of inflammatory mediators (Harlan & Liu, supra). Some of these
 mediators, such as the oxidants, can directly activate complement which
 then feeds back to further activate the neutrophils through C3a and C5a.
 This leads to further tissue damage. Intervention of this process by a
 complement inhibitory protein "homed" into the endothelial
 microenvironment by its selectin interaction, could help to stop or slow
 down this process.
 Finally, sticking of single leukocytes within the capillary lumen or
 aggregation of leukocytes within larger vessels may lead to microvascular
 occlusion and may produce ischemia. Leukocyte-mediated vascular and tissue
 injury has been implicated in the pathogenesis of a wide variety of
 clinical disorders. Inhibition of leukocyte adherence to
 endothelium-"anti-adhesion" therapy-represents a novel approach to the
 treatment of those inflammatory and immune disorders in which leukocytes
 contribute significantly to vascular and tissue injury. Studies in vitro
 indicate that close approximation of the leukocyte to the endothelial cell
 forms a protected microenvironment at the interface of the leukocyte and
 endothelial cell that is inaccessible to plasma inhibitors. Highly
 reactive oxidants, proteases, and phospholipase products released by
 adherent leukocytes at the interface can react with and damage the
 endothelium. Inhibition of such firm adherence prevents formation of a
 protected microenvironment, and thereby reduces this type of "innocent
 bystander" injury to endothelium. Inhibition of leukocyte adherence to
 endothelium will also prevent emigration to tissue, and, consequently,
 reduce tissue damage produced by emigrated leukocytes. Finally, inhibition
 of leukocyte adherence to endothelium or homotypic aggregation will
 prevent microvascular occlusion.
 The pharmaceutical compositions of the present invention can be used to
 block or inhibit cellular adhesion associated with a number of disorders.
 For instance, a number of inflammatory disorders are associated with
 selectins expressed on vascular endothelial cells and platelets. The term
 "inflammation" is used here to refer to reactions of both the specific and
 non-specific defense systems. A specific defense system reaction is a
 specific immune system reaction to an antigen. Example of specific defense
 system reactions include antibody response to antigens, such as viruses,
 and delayed-type hypersensitivity. A non-specific defense system reaction
 is an inflammatory response mediated by leukocytes generally incapable of
 immunological memory. Such cells include macrophages, eosinophils and
 neutrophils. Examples of non-specific reactions include the immediate
 swelling after a bee sting, and the collection of PMN leukocytes at sites
 of bacterial infection, e.g., pulmonary infiltrates in bacterial
 pneumonias and pus formation in abscesses).
 Additionally, the pharmaceutical compositions of the present invention can
 be used to eliminate or block the complement injury occurring in
 transplanted organs. Organs prepared for transplant can be perfused with
 the compositions of the present invention. Alternatively, organs for
 transplantation may be stored in solutions containing the compositions of
 the present invention. Such storage can occur during, for instance,
 transportation. In a further embodiment, the compositions may be used to
 flush the area from which transplant organs are removed, as from a
 cadaver. Subsequent perfusion and/or storage are also envisioned.
 Other treatable disorders include, e.g., rheumatoid arthritis,
 post-ischemic leukocyte-mediated tissue damage (reperfusion injury),
 frost-bit injury or shock, acute leukocyte-mediated lung injury (e.g.,
 adult respiratory distress syndrome), asthma, traumatic shock, septic
 shock, nephritis, vasculitis and acute and chronic inflammation, including
 atopic dermatitis, psoriasis, and inflammatory bowel disease. Various
 platelet-mediated pathologies such as atherosclerosis and clotting can
 also be treated. In addition, tumor metastasis can be inhibited or
 prevented by inhibiting the adhesion of circulating cancer cells. Examples
 include carcinoma of the colon and melanoma. In these embodiments, the
 complement moiety portion of the compositions act almost as a carrier
 protein.
 Compositions of the invention find particular use in treating the secondary
 effects of septic shock or disseminated intravascular coagulation (DIC).
 Leukocyte emigration into tissues during septic shock or DIC often results
 in pathological tissue destruction. Furthermore, these patients may have
 widespread microcirculatory thrombi and diffuse inflammation. The
 therapeutic compositions provided herein inhibit leukocyte emigration at
 these sites and mitigates tissue damage.
 The inhibitors of selectin-ligand interaction, coupled with anti-complement
 action, also are useful in treating traumatic shock and acute tissue
 injury associated therewith. Because the selectins play a role in
 recruitment of leukocytes to the sites of injury, particularly ELAM-1 in
 cases of acute injury and inflammation, inhibitors thereof may be
 administered locally or systemically to control tissue damage associated
 with such injuries. Moreover, because of the specificity of such
 inhibitors for sites of inflammation, e.g., where ELAM-1 receptors are
 expressed, these compositions will be more effective and less likely to
 cause complications when compared to traditional anti-inflammatory agents.
 The compositions of the invention can be administered to a subject in need
 thereof to treat the subject by either prophylactically preventing a
 disease state or relieving it after it has begun. The pharmaceutical
 compositions of the invention may be administered in any suitable manner,
 including parental, topical, oral, or local (such as aerosol or
 transdermal) or any combination thereof. The compositions are preferably
 administered with a pharmaceutically acceptable carrier, the nature of the
 carrier differing with the mode of administration, for example, oral
 administration, usually using a solid carrier and I.V. administration a
 liquid salt solution carrier.
 The compositions of the present invention include pharmaceutically
 acceptable components that are compatible with the patient and the protein
 and carbohydrate moieties of the compositions of the invention. These
 generally include suspensions, solutions and elixirs, and most especially
 biological buffers, such as phosphate buffered saline, saline, Dulbecco's
 Media, and the like. Aerosols may also be used, or carriers such as
 starches, sugars, microcrystalline cellulose, diluents, granulating
 agents, lubricants, binders, disintegrating agents, and the like (in the
 case of oral solid preparations, such as powders, capsules, and tablets).
 As used herein, the term "pharmaceutically acceptable" preferably means
 approved by a regulatory agency of the Federal or a state government or
 listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia
 for use in animals, and more particularly in humans.
 The formulation of choice can be accomplished using a variety of the
 aforementioned buffers, or even excipients including, for example,
 pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,
 sodium saccharin cellulose, magnesium carbonate, and the like. "Peglation"
 of the compositions may be achieved using techniques known to the art (see
 for example International Patent Publication No. WO92/16555, U.S. Pat. No.
 5,122,614 to Enzon, and International Patent Publication No. WO92/00748).
 Oral compositions may be taken in the form of solutions, suspensions,
 tablets, pills, capsules, sustained release formulations, or powders.
 Particularly useful is the administration of the compositions directly in
 transdermal formulations with permeation enhancers such as DMSO. Other
 topical formulations can be administered to treat dermal inflammation.
 A sufficient amount bf the compositions of the invention should be
 administered to the patient to ensure that a substantial portion of the
 selectin ligand expected to cause or actually causing inflammation is
 regulated, as well as to ensure that an optimal concentration of the
 complement moiety is also delivered to the site, to combat inappropriate
 complement-related activity. In this way, inflammation can either be
 prevented or ameliorated. The selection of compositions, frequency of
 administration, and amount of composition so administered will be in
 accordance with the particular disease being treated and its severity, the
 overall condition of the patient, and the judgement of the treating
 physician. Typical dosing regions will be analogous to treatment of these
 disease states by the use of antibodies and other biologicals. Typically,
 the compositions of the instant invention will contain from about 1% to
 about 95% of the active ingredient, preferably about 10% to about 50%.
 Preferably, the dosing will be between about 1-10 mg/kg. About 1 mg to
 about 50 mg will be administered to a child, and between about 25 mg and
 about 1000 mg will be administered to an adult. Other effective dosages
 can be readily determined by one of the ordinary skill in the art through
 routine trials establishing dose response curves.
 In determining the dosage of compositions to be administered, it must be
 kept in mind that one may not wish to completely block all of the selectin
 receptors, or may wish to completely block such receptors for only a
 limited amount of time (i.e. only a few hours postischemic event). In
 order for a normal healing process to proceed, at least some of the white
 blood cells or neutrophils must be brought into the tissue in the areas
 where the wound, infection or disease state is occurring. Thus, amount of
 the composition administered as a blocking agent must be adjusted
 carefully based on the particular needs of the patient while taking into
 consideration a variety of factors such as the type of disease that is
 being treated. For example, one may never desire that the neutrophils
 reoccur in an arthritic joint, but would expect such reoccurrence at some
 point after a myocardial infarct, tissue crush injury, and the like.
 In a preferred embodiment, the present invention contemplates
 pharmaceutical compositions comprising a complement inhibitory protein
 capable of binding to a selectin. Preferably, the pharmaceutical
 composition comprises a soluble CR1 molecule comprising a selectin ligand
 such as Le.sup.x, or most preferably SLe.sup.x. In one aspect, such a
 soluble CR1 molecule has LHRs A, B, C and D. In another aspect, such a
 soluble CR1 has LHRs B, C and D.
 Accordingly, it is envisioned that the pharmaceutical compositions of the
 invention will be delivered to achieve elevation of plasma levels of the
 protein to treat diseases or disorders that involve inappropriate
 complement activity, whether or not inflammatory activity is also
 involved. Diseases or disorders involving complement that require systemic
 or circulating levels of complement regulatory proteins are detailed in
 Section 2.2; supra and in Table I that follows.
 TABLE I
 Systemic Diseases and Disorders Involving Complement
 Neurological Disorders
 multiple sclerosis
 stroke
 Guillain Barre Syndrome
 traumatic brain injury
 Parkinson's Disease
 Disorders of Inappropriate or Undesirable complement
 Activation
 hemodialysis complications
 hyperacute allograft rejection
 xenograft rejection
 interleukin-2 induced toxicity during IL-2
 therapy
 Inflammatory Disorders
 inflammation of autoimmune diseases
 Crohn's disease
 adult respiratory distress syndrome
 thermal injury including burns or frostbite
 Post-Ischemic Reperfusion Conditions
 mylyocardial infarction
 balloon angioplasty
 post-pump syndrome in cardiopulmonary bypass or
 renal bypass
 hemodialysis
 renal ischemia
 mesenteric artery reperfusion after aortic
 reconstruction
 transplant organ reperfusion
 Infectious Disease or Sepsis
 Organ Preservation
 Immune Complex Disorders and Autoimmune Diseases
 rheumatoid arthritis
 systemic lupus erythematosus (SLE)
 SLE nephritis
 proliferative nephritis
 glomerulonephritis
 hemolytic anemia
 myasthenia gravis
 In particular, those disorders with may be treated by systematic
 administration are described in section 2.2 supra. In specific
 embodiments, disorders associated with extended zones of tissue
 destruction due to burn or myocardial infarct-induced trauma, and adult
 respiratory distress syndrome (ARDS), also known as shock syndrome, can be
 treated by parenteral administration of an effective amount of a
 complement inhibitory protein comprising a selectin ligand in accordance
 with the teachings herein.
 Thus, an effective amount of a composition in accordance with the present
 invention is an amount effective to inhibit complement activity, in
 addition to its other effects.
 In a preferred embodiment, the use of a complement inhibitory protein with
 selectin binding activity should be particularly helpful in
 anti-inflammatory therapy. Since the selectin ligand will home the
 complement inhibitory protein to the site of injury, it will prevent
 neutrophil rolling. This is because the selectin ligand will bind to
 selectins on the blood vessel wall, thus preventing the adhesion of
 leukocytes, particularly neutrophils.
 For example, in a particularly preferred embodiment, by adding the
 SLe.sup.x moiety, the sCR1 activity is localized to and the site of tissue
 damage, thus potentiating its anti-C activity and also blocking
 neutrophil-endothelial cell interactions such as neutrophil rolling and
 extravasation.
 In addition, the compositions can be used in the homing of CR1 to its
 ligand (the selectins) on activated endothelium, rendering lower doses
 more efficacious as compared to administration alone of sCR1 alone or and
 its present glycoforms. Heightened persistence of the SLe.sup.x -sCR1 at
 the site of inflammation is also achieved, thereby preventing further
 activation. Early neutrophil adhesion events which depend on
 selectin/ligand interaction are also blocked. Finally, the in vivo half
 life and/or bioavailability of the sCR1 is prolonged.
 5.6. APPLICATION IN THE DIAGNOSTIC FIELD
 The compositions of the present invention can be used to constitute
 detection reagents capable of binding to released or shed, or circulating
 complexes comprising a cellular adhesion molecule. Such released, or shed
 or circulating adhesion molecules may be present a result of activation of
 a particular cell comprising a cellular adhesion molecule. It is well
 known that, for instance, L-selectins are constitute expressed on the
 surface of cells and are rapidly shed following activation (Bevilacqua, M.
 P., and Nelson, R. N. (1993) supra). Thus, selectins appear to be
 controlled by their appearance and disappearance from the surface of
 cells. Circulating receptors that are shed upon activation may be assayed
 by techniques well known to those skilled in the art. An example of such
 assays is found in International Patent Publication No. WO87/03600,
 published on Jun. 18, 1987 which is incorporated herein by reference. Such
 cellular adhesion molecules may be physically distinct from the receptors
 present on the surface of the cell as, for instance, the product of an
 alternative splicing event that results in a receptor that lacks certain
 domains necessary for attachment to the cell membrane. Alternatively, such
 receptors may be fragments or portions of the natural receptor, or may be
 associated with larger membrane fragments. Further, such receptors may be
 present on intact cells.
 The compositions of the present invention may be useful in detecting the
 presence or absence of the receptors in the circulation, as in, for
 instance, a serum sample or other sample from a patient suspected of
 expressing the receptor. Alternatively, the compositions may be detectably
 labelled and used in in vitro or in vivo diagnostic imaging for the
 presence of the cellular adhesion receptors.
 In certain inflammatory conditions such as reperfusion injury, septic
 shock, and other chronic inflammatory diseases (such as for example,
 psoriasis and rheumatoid arthritis), the inflamed endothelium participates
 in the recruitment of cells to the site of injury. Accordingly, the
 compositions and methods of the present invention are useful in detecting
 the presence or absence of such inflammatory conditions by virtue of their
 demonstrated ability to bind to the activated cells and displace or
 prevent the binding of the natural ligand. In this embodiment, the
 composition of the present invention are detectably labelled by techniques
 well known in the art.
 In a further embodiment, the compositions of the present invention are
 immobilized on a solid support and the presence or absence of certain
 cellular adhesion molecules is detected by measuring or calculating thd
 amount of binding that occurs. In this embodiment, certain monoclonal
 antibodies well known in the art may be used in conjunction with the
 compositions.
 The compositions can also be used to study inflammatory and complement
 mediated diseases or disorders by. virtue of their direct interaction with
 mediators of inflammation as described herein. In particular, the
 compositions can be used in either In vitro or in vivo methods. In in
 vitro methods the samples may be fluid specimens or tissue specimens and
 can include enzyme-linked assays, such as immunoperoxidase assays or
 staining of tissue samples.
 The compositions of the invention can be used as part of a kit, especially
 a diagnostic kit. Such a kit may include, for instance, the compositions
 of the invention, as well as, components that are detectably labelled, as
 for instance, monoclonal antibodies to the particular cellular adhesion
 molecule. In one embodiment, the kit includes one or more compositions,
 along with the appropriate dilution and incubation buffers, a detectably
 labelled binding partner suitable for use in a sandwich assay format, and
 a substrate reagent.
 6. EXAMPLE 1
 6.1. GENERATION OF A SOLUBLE DELETION MUTANT OF COMPLEMENT RECEPTOR 1
 The following experiments detail the generation of several soluble deletion
 mutants of complement receptor type 1 useful in the present invention.
 6.2 GENERATION OF A SOLUBLE DELETION MUTANT OF CR1 (SCR1[DES-A]) LACKING
 LHR-A
 Plasmid pBSABCD is described in International Patent Publication No.
 WO89/09220 "The Human C3b/C4b Receptor (CR1)" by Fearon, D. T., et al.,
 published Oct. 5, 1989; (see also, Klickstein, L. B., et al., (1988) J.
 Exp. Med 168:1699-1717). This plasmid harbors a full-length cDNA for human
 CR1 inserted as a 6.86-kilobase (kb) EcoRI-Eco-RV piece in the EcoRI-SmaI
 sites of pBluescript KS+ (Stratagene, La Jolla, Calif.); thus, the EcoRV
 and SmaI sites did not regenerate. pBSABCD was further modified by
 introducing a translational stop codon at the junction of the
 extracellular and transmembrane regions to yield pBL-sCR1 capable of
 expressing a soluble CR1 protein lacking the transmembrane and cytoplasmic
 domains. (International Patent Publication No. WO89/09220 "The Human
 C3b/C4b Receptor (CR1)" by Fearon, D. T., et al., published Oct. 5, 1989;
 Weisman, H. F., et al., (1990) Science 249:146-151).
 pBL-SCR1 was digested with ClaI and BalI, and the resulting fragments (3.96
 and 5.9 kb) were purified from low melting temperature agarose gel.
 Plasmid pBR322 was digested with ClaI and BalI, and the 2.9-kb fragment
 was purified from agarose gel and ligated to the 5.9-kb fragment from
 pBL-sCR1. The ligation mix was transformed into competent E. coli
 DH5.alpha. cells (GIBCO BRL), and the resulting plasmid, pBR8.8, was
 purified and digested with XbaI, generating two fragments of 7.45 and 1.35
 kb. The 7.45-kb fragment was purified and religated into a circular form.
 The resulting plasmid, pBR7.45, was digested with ClaI and BalI, and the
 4.5-kb fragment containing the CR1 cDNA was ligated to the 3.96-kb
 fragment from pBL-sCR1 generating pBL-sACD lacking LHR-B.
 Digestion of pBL-CR1c2, also referred to as pBL-sACD (Makrides et al.,
 (1992) J. Biol. Chem. 267:24754-24761) with NarI and NsiI removed 76 bp
 from the 3' end of the leader, the entire LHR-A, and 57 bp from the 5' end
 of LHRC; the 7.07 kb fragment was purified from agarose gel and ligated to
 two synthetic double-stranded oligonucleotides (Operon Technologies,
 Alameda, Calif.), 68 and 66 bp in length having the following sequence:
 1. 5'- CG CCC GGT CTC CCC TTC TGC TGC GGA GGA TCC
 3'- GGG CCA GAG GGG AAG ACG ACG CCT CCT AGG
 CTG CTG GCG GTT GTG GTG CTG CTT GCG GTG
 GAC GAC CGC CAA CAC CAC GAC GAA CGC GAC
 CCG GTG -3' [SEQ ID NO. 1]
 GGC CAC CGG ACC -5' [SEQ ID NO. 2]
 2. 5'- GCC TGG GGT CAA TGT CAA GCC CCA GAT CAT
 3'- CCA GTT ACA GTT CGG GGT CTA GTA
 TTT CTG TTT GCC AAG TTG AAA ACC CAA ACC
 AAA GAC AAA CGG TTC AAC TTT TGG GTT TGG
 AAT GCA -3' [SEQ ID NO. 3]
 TT -5' [SEQ ID NO. 4]
 (Operon Technologies, Alameda, Calif.). These oligonucleotides restored the
 missing sequences from both the leader and LHR-C, respectively. In
 addition, a single nucleotide change was designed in one of the
 oligonucleotides, such that the first codon of LHR-C in SCR 15 coded for
 glutamine, instead of the native histidine. The rationale for this
 modification was two-fold: (1) to ensure that the junction between the
 leader peptide and the coding region of the mature protein would be the
 same as in the native sCR1 (i.e. Glycine/Glutamine) thus avoiding
 potential difficulties with cleavage of the leader by signal peptidases;
 (2) to ensure that the N-terminal amino acid in the processed protein
 would be the same as in the native CR1 (Klickstein et al., (1988) J. Exp.
 Med. 168: 1699-1717) thus minimizing the potential for immunogenicity. The
 ligation mix was transformed into Escherichia coli strain DH5.alpha.
 (Gibco BRL, Gaithersburg, Md.) to produce plasmid pBL-CR1c8 containing the
 leader, LHR-C and LHR-D.
 pBL-CR1c8 was linearized with NsiI and dephosphorylated using bacterial
 alkaline phosphatase (Gibco BRL) according to the manufacturer's
 instructions. pBL-CR1c, also referred to as pBL-sCR1 [Weisman et al.,
 (1990) Science 249:146-151] was digested with NsiI and the 1.35 kb
 fragment containing most of LHR-B and the first 56 nucleotides from LHR-C
 was purified from agarose gel, and ligated to the linearized pBL-CR1c8.
 This effected the assembly of pBL-CR1c6A containing LHRs B, C, and D. The
 correct orientation of the BCD insert was determined by restriction
 digestion analysis.
 The insert was excised by digestion with XhoI, and purified from agarose
 gel. The expression plasmid pTCSgpt (International Patent Publication No.
 WO89/09220 "The Human C3b/C4b Receptor (CR1)" by Fearon, D. T. et al.,
 published Oct. 5, 1989; Carson et al., (1991) J. Biol. Chem. 266:
 7883-7887) was digested with XhoI, dephosphorylated using bacterial
 alkaline phosphatase, and ligated to the BCD fragment. The ligation mix
 was transformed into E. coli DH1, generating plasmid pT-CR1c6A. The
 correct insert orientation was determined by BglI restriction digestion,
 and pT-CR1c6A was prepared on large scale. pT-CR1c6A is a plasmid which
 harbors the coding sequence for the soluble deletion mutant of CR1 lacking
 the LHR-A as well as the transmembrane and cytoplasmic domains. The
 resulting soluble deletion mutant is termed sCR1[des-A] containing LHR's
 B, C, and D.
 7. EXAMPLE 2
 7.1 CONSTRUCTION OF A SOLUBLE DELETION MUTANT OF CR1 CONTAINING SCR'S 15-18
 A DNA fragment composed of the CR1 leader and Short Consensus Repeats (SCR)
 15 through 18 was PCR-synthesized using pBL-CR1c8 as template [Makrides et
 al. (1992) J. Biol. Chem. 267, 24754-24761]. The 5' "sense" primer
 hybridized to the pBluescript polylinker region upstream of the CR1
 leader, and contained an XhoI restriction site, underlined:
 5'-CCCCCCCTCGAGGTCGACGGTATCGATAAGC-3' [SEQ ID NO. 5]
 The 3' "antisense" primer contained restriction enzyme recognition
 sequences for BglII and NotI sites, underlined:
 5'-TATCAAATGCGGCCGCTAAGAATACCCTAGATCTGGAGCAGCTTGGTAACTCTGGC-3' [SEQ ID NO.
 6]
 The resulting 980-bp fragment was digested with XhoI and NotI, and ligated
 into pBluescript KS(+) (Stratagene, La Jolla, Calif.) previously
 restricted with XhoI and NotI. The ligation mix was transformed into E.
 coli DH5.alpha. competent cells (GibcoBRL, Gaithersburg, Md.) to yield
 plasmid pB-CR1(15-18) (3.86 kb). This was linearized at the 3' terminus of
 SCR 18 using BglII, and blunt-ended with mung bean nuclease (New England
 Biolabs, Beverly, Mass.) used according to the manufacturer's
 recommendations. The linearized plasmid was ligated to a synthetic
 double-stranded oligonucleotide (Operon Technologies, Inc., Alameda,
 Calif.) composed of the following two complementary strands:
 5'-GATGAACTAGTCTCGAGAG-3' [SEQ ID NO. 7]
 5'-CTCTCGAGACTAGTTCATC-3' [(SEQ ID NO. 8]
 The double-stranded oligonucleotide restored the missing base-pairs from
 the 3' terminus of SCR 18, and introduced a translational stop codon,
 followed by SpeI and XhoI restriction sites. The Ligation mix was
 transformed into E. coli DH5.alpha. competent cells yielding plasmid
 pB-CR1(15-18A) (3.88 kb).
 The DNA fragment composed of the CR1 leader and SCR 15-18 was excised from
 pB-CR1(15-18A) by digestion with XhoI, purified from agarose gel using the
 Geneclean Kit (BIO 101, La Jolla, Calif.), and ligated to the expression
 vector pTCSgpt [Carson et al., (1991) J. Biol. Chem. 266, 7883-7887]
 previously restricted with XhoI and dephosphorylated with calf intestinal
 alkaline phosphatase (Boehringer Mannheim, Indianapolis, Ind.).
 Transformation of the ligation mix into E. coli DH1 competent cells
 yielded plasmid pT-CR1c12 (8.52 kb).
 7.2 TRANSFECTION AND SELECTION OF STABLE CELL LINES
 pT-CR1c12 was linearized by FspI digestion, phenol-extracted,
 ethanol-precipitated and resuspended in sterile water. 30 .mu.g of
 recombinant plasmid was cotransfected with 3 .mu.g pTCSdhfr [Carson et
 al., (1991) J. Biol. Chem. 266, 7883-7887] into CHO DUKX-B11 cells
 deficient in dihydrofolate reductase [Urlaub and Chasin (1980) Proc. Natl.
 Acad. Sci. USA 77, 4216-4220] using electroporation with the Gene Pulser
 (Bio-Rad) at 960 .mu.F and 230 V. The transfected cells were transferred
 to non-selective .alpha.-Minimum Essential Medium (.alpha.-MEM)
 supplemented with 10% heat-inactivated fetal bovine serum, 1%
 penicillin-streptomycin, 50 .mu.g/ml gentamicin, 4 mM glutamine (Gibco
 BRL), 10 .mu.g/ml each of thymidine, adenosine, and deoxyadenosine (Sigma,
 St. Louis, Mo.). After two days the cells were selected in .alpha.-MEM
 supplemented with 10% dialyzed fetal bovine serum, 1%
 penicillin-streptomycin, 50 .mu.g/ml gentamicin, 4 mM glutamine, 20 mM
 HEPES pH 7.0, 6 .mu.g/ml mycophenolic acid, 250 .mu.g/ml xanthine, and 15
 .mu.g/ml hypoxanthine (Sigma). Clones secreting SCR 15-18 were identified
 by enzyme immunoassay (Cellfree.RTM. CD35; T Cell Sciences, Inc.), and the
 complement inhibitory activity of the proteins was confirmed using
 hemolytic assays [Yeh et al., (1991) J. Immunol. 146, 250-256].
 High-expressing clones were selected in growth media containing
 methotrexate (Lederle, Pearl River, N.Y.).
 8. EXAMPLE 3
 8.1 CONSTRUCTION OF A SOLUBLE DELETION MUTANT OF CR1 LACKING LHR D
 (sCR1[des-D])
 Plasmid pBSABCD was obtained from Dr. Douglas Fearon [Klickstein et al.,
 (1988) J. Exp. Med. 168, 1699-1717]. This plasmid harbors a full-length
 cDNA for human CR1 inserted as a 6.86 kilobase (kb) EcoR1-EcoRV piece in
 the EcoR1-SmaI sites of pBluescript KS+ (Stratagene, La Jolla, Calif.);
 thus, the EcoRV and SmaI sites did not regenerate. pBSABCD was further
 modified as described [Weisman et al., (1990) Science 249, 146-151] to
 yield pBL-sCR1 capable of expressing a soluble CR1 protein lacking the
 transmembrane and cytoplasmic domains.
 An unique NruI restriction site was introduced in pBL-sCR1 at position 4200
 basepair (bp), i.e., at the junction of LHR-C and -D. The enzyme site was
 engineered by site-directed mutagenesis [Kunkel (1985) Proc. Natl. Acad.
 Sci. USA 82, 488-492] using the Muta-gene Phagemid Kit (Bio-Rad
 Laboratories, Melville, N.Y.). The 40-base phosphorylated mutagenic
 oligonucleotide (New England Biolabs, Beverly, Mass.) had the following
 sequence:
 3'CGACACTTGAAAGACAAGCGCTACCAGTGACATTTTGGGG5' [SEQ ID NO. 9]
 The underlined bases are those which differ from the wild-type sequence.
 DNA templates were sequenced by the dideoxynucleotide chain termination
 method [Sanger et al., (1977) Proc. Natl. Acad. Sci. USA 74, 5463-54673]
 using the Sequenase kit (U.S. Biochemical, Cleveland, Ohio).
 The mutagenized plasmid pBL-sCR1N (9.8 kb) was digested with NruI and
 BglII, and the 7.8 kb fragment was isolated from agarose and ligated to a
 double-stranded synthetic oligonucleotide composed of the following
 complementary strands:
 5'-CGCTTAAGCTCGA-3' [SEQ ID NO. 10]
 5'-GATCTCGAGCTTAAGCG-3' [SEQ ID NO. 11]
 The double-stranded synthetic oligonucleotide restored the missing base
 pairs from the 3' terminus of SCR 21 (LHR C), and introduced a
 translational stop codon followed by XhoI and BglII restriction sites. The
 resulting plasmid pBL-CR1c7 (7.8 kb) was digested with XhoI, and the
 insert was ligated into the expression vector pTCSgpt [Carson et al.,
 (1991) J. Biol. Chem. 266, 7883-7887] previously restricted with XhoI and
 dephosphorylated with bacterial alkaline phosphatase (GibcoBRL,
 Gaithersburg, Md.) used according to the manufacturer's recommendations.
 Transformation of the ligation mix into E. coli DH1 competent cells
 yielded plasmid pT-CR1c7.
 Transfection and Selection of Stable Cell Lines.
 pT-CR1c7 was linearized by FspI digestion, phenol-extracted,
 ethanol-precipitated and resuspended in sterile water. 30 .mu.g of
 recombinant plasmid was cotransfected with 3 .mu.g pTCSdhfr [Carson et
 al., (1991) J. Biol. Chem. 266, 7883-7887] into CHO DUKX-B11 cells
 deficient in dihydrofolate reductase [Urlaub and Chasin (1980) Proc. Natl.
 Acad. Sci. USA 77, 4216-4220] using electroporation with the Gene Pulser
 (Bio-Rad) at 960 .mu.F and 230 V. The transfected cells were transferred
 to non-selective .alpha.-Minimum Essential Medium (.alpha.-MEM)
 supplemented with 10% heat-inactivated fetal bovine serum, 1%
 penicillin-streptomycin, 50 .mu.g/ml gentamicin, 4 mM glutamine (Gibco
 BRL), 10 .mu.g/ml each of thymidine, adenosine, and deoxyadenosine (Sigma,
 St. Louis, Mo.). After two days the cells were selected in .alpha.-MEM
 supplemented with 10% dialyzed fetal bovine serum, 1%
 penicillin-streptomycin, 50 .mu.g/ml gentamicin, 4 mM glutamine, 20 mM
 HEPES pH 7.0, 6 .mu.g/ml mycophenolic acid, 250 .mu.g/ml xanthine, and 15
 .mu.g/ml hypoxanthine (Sigma). Clones secreting sCR1[desD] LHR's A, B, and
 C were identified by enzyme immunoassay (Cellfree.RTM. CD35; T Cell
 Sciences, Inc.), and the complement inhibitory activity of the proteins
 was confirmed using hemolytic assays [Yeh et al., (1991) J. Immunol. 146,
 250-256]. High-expressing clones were selected in growth media containing
 methotrexate (Lederle, Pearl River, N.Y.).
 9. EXAMPLE 4
 9.1 GENERATION OF SOLUBLE CONSTRUCTS OF CR1 CONTAINING THE SLEX
 CARBOHYDRATE MOIETY
 Any of the foregoing soluble deletion mutants of CR1 or other complement
 moiety as defined herein can be manipulated to contain a carbohydrate
 moiety useful within the scope of the present invention. The following
 examples describe the generation of sCR1[des-A]sLe.sup.x a soluble
 deletion mutant of CR1 lacking LHR-A and containing the sLex carbohydrate
 moiety.
 9.1.1. TRANSFECTION OF THE sCR1[des-A] CONSTRUCT INTO LEC-11 CELLS
 Following linearization by FspI restriction digestion, pT-CR1c6A (Example
 1, Section 6.2, Supra) was cotransfected into LEC11 cells (Campbell, C.,
 and Stanley, P., 1984, J. Biol. Chem. 259:11208-11214) with
 FspI-linearized pTCSdhfr* containing an altered mouse dihydrofolate
 reductaise, cDNA that displays an abnormally low affinity for methotrexate
 (Simonsen, C. C. and Levinson, A. D. (1983) Proc. Natl. Acad. Sci. USA 80:
 2495-2499). Clones secreting sCR1[desA]sLe.sup.x were identified by enzyme
 immunoassay (Cellfree CD35; T Cell Diagnostics, Inc.), and the complement
 inhibitory activity of the protein was confirmed using hemolytic assays
 (Yeh et al., (1991) J. Immunol. 146: 250-256). High-expressing clones were
 selected in growth media containing methotrexate (Lederle, Pearl River,
 N.Y.).
 9.1.2 Cell Culture Droduction of sCR1[des-A]sLe.sup.x.
 CHO DUKX-B11 cells secreting sCR1[desLHR-A] or CHO LEC-11 cells secreting
 sCR1[desLHR-A]sLe.sup.x were grown in T-225 flasks in 1:1 Dulbecco's
 modified Eagle's medium with high glucose/Ham's nutrient mixture F12
 without hypoxanthine and thymidine (JRH Biosciences, Lenexa, Kans.)
 supplemented with 2.5% heat-inactivated fetal bovine serum (Hyclone,
 Logan, Utah). The pH of the media was adjusted to 7.8 to using sodium
 bicarbonate to minimize sialidase activity present in the conditioned
 medium. The conditioned medium from these cultures was harvested three
 times a week by decanting, filtered, and frozen at -70.degree. C. until
 purification. Productivity was monitored by ELISA.
 9.1.3 Purification of sCR1[desA]sLe.sup.x.
 Filtered cell culture supernatants containing sCR1[desA]sLe.sup.x or
 SCR1[des-A] were buffer exchanged and concentrated by cross-floaw
 ultrafiltration (30,000 molecular weight cut-off), filtered again, applied
 to a S-Sepharosa Fast Flow cation exchange column, and eluted with a high
 salt concentration (0.5 M sodium chloride). The cation exchange eluant was
 precipitated with ammonium sulfate, separated by centrifugation,
 resuspended in PBS, and filtered. The filtrate was adjusted to 0.8 M
 ammonium sulfate, loaded on a Butyl-Toyopearl 650M column and eluted with
 a step to 0.09 M ammonium sulfate. The eluant was concentrated using
 Centriprep-30 concentrators (Amicon), subjected to size exclusion
 chromatography on a Toyopearl HW55F column, again concentrated using
 Centriprep-30 concentrators, sterile filtered, and stored frozen at
 -70.degree. C. The purification process was monitored by absorbance at 280
 nm and by ELISA. Protein purity was examined by SDS-PAGE with either
 Coomassie Blue or silver staining and scanning densitometry. Endotoxin
 levels were determined using the Limulus Amebocyte Lysate assay
 (Associates of Cape Cod, Inc., Woods Hole, Mass.).
 10. EXAMPLE 5
 10.1 IN VITRO COMPLEMENT REGULATORY ACTIVITY OF SCR1[des-A]SLEX AND
 SCR1[des-A]
 The in vitro regulatory activities of sCR1[des-A]sLe.sup.x were compared to
 those of sCR1[des-A], which is the same protein except lacking sLe.sup.x
 glycosylation and which has been shown to selectively inhibit alternative
 complement activation in vitro. sCR1[desA]sLe.sup.x was constructed and
 expressed in LEC11 cells, and purified from cell culture supernatants as
 described in the previous examples. sCR1[des-A] was constructed as
 described above and expressed in CHO DUKX-B11 cells as described above for
 sCR1[des-A]sLex except that pT-CR1c6a was cotransfected into DUKX-B11 with
 FspI-linearized pTCSdhfr. sCR1[des-A] was purified from cell culture
 supernatants as described above for sCR1[des-A]sLex.
 sCR1[desA]sLe.sup.x and sCR1[des-A] competed equally for the binding of
 dimeric C3b to erythrocyte CR1. sCR1[des-A]sLe.sup.x and sCR1[des-A] were
 equivalent in their capacity to serve as a cofactor in the factor I
 mediated degradation of the C3b .alpha.-chain. sCR1[des-A]sLe.sup.x and
 sCR1[des-A] were equivalent in their capacity to inhibit alternative
 complement mediated erythrocyte lysis using C4-deficient guinea pig serum
 as a complement source. sCR1[des-A]sLe.sup.x and sCR1[des-A] were
 equivalent inhibitors of complement mediated erythrocyte lysis under
 conditions which allow classical pathway activation. Both, however, were
 significantly less effective inhibitors of classical pathway mediated
 hemolysis than sCR1, a soluble recombinant protein containing the entire
 extracellular sequence of CR1. Thus, sCR1[desA]sLe.sup.x, like
 sCR1[des-A], is a selective inhibitor of alternative complement pathway in
 vitro.
 10.1.1 Complement Proteins and Antibodies.
 Human C4, C3, C3b, and chemically cross-linked dimeric C3b (C3b2) were
 prepared as described previously (Makrides et al., 1992, Scesney et al.,
 Eur. J. Immunol., 1996, 26:1729-1735). C4 was treated with methylamine to
 produce C4ma, a C4b-like form of the protein (Makrides et al., 1992; Law
 and Levine, 1980). C3b, C3b.sub.2, and C4ma were radiolabeled with
 .sup.125 I using Iodo-beads (Pierce Chemical Co.) according to the
 manufacturer's recommendations. C4 deficient guinea pig serum was obtained
 commercially (Sigma).
 10.1.2 C3b.sub.2 Binding Studies.
 The binding of sCR1[desA]sLe.sup.x and sCR1[des-A] to .sup.125 I-C3b.sub.2
 was assessed by competition with native CR1 on human erythrocytes (Weisman
 et al., 1990; Makrides et al., 1992). Human erythrocytes were diluted with
 an equal volume of Alsever's solution (113 mM dextrose, 27 mM sodium
 citrate, 2.6 mM citric acid, 72 mM sodium chloride, pH 7) and stored at
 4.degree. C. until use. There was no difference in C3b.sub.2 binding to
 freshly drawn erythrocytes and those stored in Alsever's solution.
 Immediately prior to use, erythrocytes were washed three times with PBS,
 0.1% BSA, and 0.01% sodium azide. .sup.125 I-C3b.sub.2 (0.55 nM) was
 incubated with erythrocytes (4.times.10.sup.9 cells/ml) for 60 min on ice
 (0.degree. C.) in the presence of varying concentrations of sCR1,
 sCR1[des-A], sCR1[des-A]sLex C3b.sub.2, or C3b. Bound and free .sup.125
 I-C3b.sub.2 were separated by centrifugation through dibutyl phthalate.
 Nonspecific binding was determined in the presence of 0.71 mg/ml purified
 rabbit anti-sCR1 antibody.
 RESULTS
 C3b.sub.2 Binding Studies.
 The competition of sCR1[des-A], sCR1[des-A]sLex and sCR1 with .sup.125
 I-C3b.sub.2 binding to erythrocyte CR1 was assessed. From the
 concentration of competitor required to inhibit maximal .sup.125
 I-C3b.sub.2 binding by 50%, apparent dissociation constants (K.sub.d,app)
 for sCR1, C3b.sub.2, and C3b were estimated to be 2.times.10.sup.-9 M,
 3.times.10.sup.-8 M, and 6.times.10.sup.-7 M, respectively, values which
 are similar to results obtained in earlier studies (Weisman et al., 1990;
 Wong and Farrel, 1991; Makrides et al., 1992). sCR1[des-A]sLe.sup.x or
 sCR1[des-A] compete equally for .sup.125 I-C3b.sub.2 binding to
 erythrocyte CR1.
 10.1.3 Cofactor Activity for Proteolysis of Fluid Phase C3b or C4ma by
 Factor I.
 The capacity of sCR1[desA]sLe.sup.x or sCR1[des-A] to promote the specific
 proteolysis of the C3b or C4ma .alpha.-chain was assessed on SDS-PAGE
 (Wong et al., 1985; Weisman et al., 1990; Scesney et al., Eur. J.
 Immunol., 1996, 26:1729-1735). .sup.125 I-C3b (6.8.times.10.sup.-9 M) or
 .sup.125 I-C4ma (5.6.times.10.sup.-8 M) was incubated in PBS with factor I
 (0.25 .mu.M) and varying concentrations of either sCR1[des-A]sLe.sup.x or
 sCR1[des-A] for 20 min at 37.degree. C. followed by 5 min on ice
 (0.degree. C.). Under these conditions the proteolysis of the C4ma C3b
 .alpha.-chain was dependent on the concentration of cofactor. The
 remaining intact C3b .alpha.-chain was separated on reduced SDS-PAGE and
 the bands were cut out and measured in a .gamma.-counter.
 RESULTS
 Cofactor Activity for the Factor I Proteolysis of the C3b .alpha.-chain and
 of the C4ma .alpha.-chain.
 The specific proteolysis of .sup.125 I-C3b or .sup.125 I-C4ma by factor I
 was monitored on SDS-PAGE under conditions in which the extent of
 .alpha.-chain cleavage was dependent on the concentration of cofactor,
 either sCR1[des-A]sLe.sup.x or sCR1[des-A]. The loss of the band
 representing the intact C3b .alpha.-chain required similar concentrations
 of either sCR1[des-A]sLe.sup.x or sCR1[des-A]. The loss of the band
 representing the intact C4ma .alpha.-chain also required similar
 concentrations of either sCR1[des-A]sLe.sup.x or sCR1[desLHR-A]. It can be
 concluded that sCR1[des-A]sLe.sup.x and sCR1[des-A] were equivalent in
 their capacity to serve as a cofactor in the Factor I mediated degradation
 of the C3b .alpha.-chain.
 10.1.4 Hemolytic Assay for Inhibition of Classical and of Alternative
 Complement Activation.
 The inhibition of complement activation was assessed as previously
 described (Weisman et al., 1990; Yeh et al., 1991). Sheep erythrocytes
 sensitized with rabbit anti-sheep erythrocyte antibodies (Diamedix, Miami,
 Fla.) were lysed using human serum as a complement source in 100 mM HEPES,
 150 mM sodium chloride, 0.1% BSA, pH 7.4. Sensitized sheep erythrocytes
 (10.sup.7 cells/ml), normal human serum (1 in 400 dilution), and varying
 concentrations of sCR1[des-A] or sCR1[des-A]sLe.sup.x were incubated for
 60 min at 37.degree. C. in V-bottom microtiter plates, the cells pelleted
 by centrifugation, and the supernatants transferred to a flat bottom
 microtiter plate and the absorbance at 405 nm determined in order to
 guantitate released hemoglobin. Samples were paired with identical
 controls lacking human serum (complement-independent lysis). Both samples
 and controls were run in triplicate. Control values were subtracted from
 sample values and the fractional inhibition was determined relative to the
 uninhibited (no added sCR1[des-A]sLe.sup.x or sCR1[des-A]) sample.
 The inhibition of alternative pathway hemolysis was assessed using the
 modified method of Platts-Mills and Ishizaka (1974). Rabbit erythrocytes
 were lysed using C4 deficient guinea pig serum as complement in 100 mM
 HEPES, 0.15 N sodium chloride, 0.1% bovine serum albumin, pH 7.4 with
 added EGTA and Mg.sup.2+ to 8 mM and 5 mM, respectively. Rabbit
 erythrocytes (1.2.times.10.sup.7 cells/ml), C4 deficient guinea pig serum
 (1 in 8 dilution), and sCR1[des-A]sLe.sup.x or sCR1[des-A] were incubated
 60 min at 37.degree. C. in a V-bottom microtiter plate, and released
 hemoglobin was determined as before.
 Inhibition of Heinolysis by the Alternative Complement Pathway Using
 C4-deficient Guinea Pig Serum.
 To rule out interference from either pre-existing or newly generated C4b,
 the alternative pathway lysis of rabbit erythrocytes was examined using
 C4-deficient guinea pig serum as a complement source. Equivalent
 concentrations of sCR1, sCR1[des-A], or sCR1[desA]sLe.sup.x were required
 to inhibit alternative complement-mediated erythrocyte lysis.
 Inhibition of Hemolysis Initiated by the Classical Complement Pathway.
 The inhibition of complement lysis of antibody-sensitized sheep
 erythrocytes required approximately equivalent concentrations of
 sCR1[desA]sLe.sup.x or sCR1[desLHR-A]. These concentrations, however, were
 approximately 50-fold higher than those required for inhibition by sCR1
 which contains LHR's A, B, C, and D.
 11. EXAMPLE 6
 11.1 ANALYSIS of sCR1[des-A]sLex
 In this Example the the purified proteins sCR1[des-A] and sCR1[des-A]sLex
 are compared in Western blot analysis.
 11.1.1 ANTIBODIES
 sCR1 was prepared as previously described (Weisman et al., 1990; Yeh et
 al., 1991). Polyclonal rabbit anti-sCR1 antibodies were prepared and
 purified as described (Makrides et al., 1992). CSLEX-1 (anti-sialyl
 Lewis.sup.x) was obtained from Becton Dickinson. FH6 (anti-sialyl
 di-Le.sup.x) was obtained from Dr. S. Hakomori (Biomembrane Institute,
 Seattle Wash.). DREG-56 (anti-E-selectin) was obtained from Endogen,
 Cambridge, Mass.; anti-CD15 (anti-Lex)was obtained from AMAC (Westbrook,
 Me.
 11.1.2 Western Blot Analysis of sCR1[desA]sLe.sup.x.
 Western Blot analysis was conducted acccording to the following procedure:
 a) glycoproteins obtained from the transfection of LEC-11 cells with the
 sCR1[des-A] construct described above were run subjected to SDS
 polyacrylamide gel electrophoresis under reducing and non-reducing
 conditions with appropriate controls and standards.
 b) the glycoprotein bands were transferred to solid support membranes
 (Immobilon.TM.) via semi-dry electrophoretic transfer (Integrated
 Separation Systems)
 c) the membranes containing the transferred glycoproteins were blocked with
 1% non-fat dry milk proteins for 2 hrs., (or overnight) or other blocking
 reagents such as bovine serum albumin (at about 2.0%) and gelatin (at
 about 0.3%). The latter blocking reagents are preferable to avoid
 complications due to potential SLe.sup.x glycosylated proteins present in
 the milk solution (such as IgA).
 d) the replicate membranes were then reacted with antibodies to CR1 and/or
 sCR1[des-A], antibodies to Lex(DAKO, AMAC, CD15), antibodies to SLe.sup.x
 (CSLEX-1, available from ATCC, and from Becton Dickinson), and isotype
 matched control antibodies for 2hrs.
 e) the membranes were washed in wash buffer(PBS-tween) 4 times for about
 10-15 minutes each.
 f) the membranes were then reacted with HRP-labelled anti-murine antibodies
 or anti-rabbit antibodies (all available from various vendors such as
 Bio-rad, Southern Biotech, Tago) for 2 hrs.
 g) the membranes were washed as in (e).
 h) the membranes are developed with HRP subtrate (Bio-rad, Sigma) to
 visualize the glycoprotein bands reactive with each primary antibody, or,
 a chemilumincescent method referred to as "ECL" (Enhanced
 Chemiluminescence, Amersham).
 RESULTS
 11.2 Western Blot Analysis.
 The above-described technique using the ECL Western Blot procedure from
 Amersham and antibodies to SLe.sup.x (CSLEX1) and antibodies to CR1
 (rabbit polyclonal antibodies) was performed using the material derived
 from LEC-11 cells transfected with the complement moiety termed
 sCR1[des-A] obtained through the method described above to yield
 sCR1[desA] sLe.sup.x. The results of this Western Blot analysis clearly
 demonstrated that sCR1[desA]sLe.sup.x derived from LEC-11 cells (Pamela
 Stanley, Albert Einstein College of Medicine) bears sLe.sup.x moieties as
 determined by staining with CSLEX1 antibodies, (ATCC HB 8580, see U.S.
 Pat. No. 4,752,569) while material derived from transfection of the
 sCR1[des-A] construct into DUKX.B11 CHO cells does not.
 FIG. 1B shows the results of this analysis, the first lane with material in
 it contains molecular weight standards. The second lane contains lysate
 derived from HL-60 cells (positive control for CSLEX1 mAb). The third lane
 contains sCR1[des-A] material derived from DUKX.B11 CHO cells and the
 fourth lane contains sCR1[des-A]sLe.sup.x material derived from LEC-11
 cells. Of the two lanes containing the sCR1[des-A] material, only that
 lane derived from LEC-11 cells (Lane 4) was identified by the CSLEX1 mAb
 as demonstrated by two clear bands consistent with two glycosylation forms
 of sCR1[des-A]. Both lanes containing sCR1[des-A] (Lane 3 from DUX.B11,
 and Lane 4 from LEC-11) reacted with a polyclonal antibody to sCR1[des-A]
 as expected (FIG. 1C). FIG. 1A is a coomasie-blue stained SDS-PAGE gel in
 the same material.
 11.3 SECOND WESTERN BLOT ANALYSIS
 In a separate experiment sCR1[desLHR-A] and sCR1[desA]sLe.sup.x were
 subjected to SDS-PAGE using a 4-20% gel (ISS) and non-reducing conditions.
 The gels were blotted onto a membrane (Immobilon-P) using a semi-dry
 transblotting apparatus (ISS). The membranes were blocked overnight at
 room temperature in a solution of tris buffered saline (TBS) containing 2%
 BSA, 1% normal goat serum, 0.05% sodium azide. The blot was probed with
 FH6 (anti-sialyl di-Le.sup.x, Hakomori supernatant, diluted 1:1 in TBS
 blocking buffer) for 2 h at room temperature. After extensive washing in
 PBS with 0.05% Tween-20, the blot was probed with horseradish peroxidase
 (HRP) conjugated goat anti-mouse IgM (Tago, 1 ug/ml in blocking buffer)
 for 1 h at room temperature. After extensive washing in PBS with 0.05%
 Tween-20, the blot was incubated with a chemi-luminescent substrate (ECL
 kit, Amersham) for 1 minute, exposed to x-ray film for 30-120 s, and the
 film developed. The blot was then stripped and re-probed with rabbit
 polyclonal anti-sCR1 (1:2000 in blocking buffer) for 1 h, washed
 extensively, probed with HRP conjugated anti-rabbit Ig (Amersham), washed
 and detected as before.
 RESULTS OF THE SECOND WESTERN BLOT ANALYSIS
 In the second western blot analysis both CSLEX-1, a monoclonal antibody
 that reacts with sLe.sup.x oligosaccharides, and FH60, a monoclonal
 antibody that reacts with sialyl di-Le.sup.x, bound to sCR1[desA]sLe.sup.x
 but not to sCR1[desA]. Both oligosaccharide structures have been shown to
 be ligands for selectins (Goelz, S. et al., J. Biol. Chem. (1994)
 269:1033-1040). Parekh et al. (1992) J. Biochem. (Tokyo) 16d,137,
 identified the carbohydrate strucures responsible for binding to an
 E-selectin affinity column to be sialyl di-Le.sup.x. No insight into the
 number of N-linked glycosylation sites used, or how many of those
 terminate in sLe.sup.x, can be derived from this experiment.
 FIGS. 2A through 2C detail the results of the second Western blot
 experiment. FIGS. 2A through 2C are an analysis of the same polyacrylamide
 gel. In lane 1 of each Figure are the molecular weight standards. Lane 2
 of each Figure is the purified sCR1[des-A] material obtained from DUKX-B11
 cells. Lane of 3 each is an irrelevant control material. Lanes 4, 5 and 6
 of each gel are sCR1[des-A]sLex at varying stages during the purification
 procedure.
 FIG. 2A is a coomassie blue stained polyacrylamide gel pattern. The
 predominant bands at approximately 187 kd in lanes 2, and 4-6 are the
 sCR1[des-A] protein, lane 2 obtained from DUKX-B11 cells and lanes 4-6
 obtained from LEC-11 cells. FIG. 2B is the same gel as FIG. 2 Western
 blotted and probed with and anti-sCR1[des-A] polyclonal serum. As
 expected, all lanes containing sCR1-[des-A], whether derived from DUKX-B11
 cells or LEC-11 cells are positive for sCR1[des-A]. FIG. 2C is the same
 blot as FIG. 2B stripped and reprobed with an antibody specific for the
 sialyl di-Lewis x antigen represented by the shorthand notation
 NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.beta.1-3Gal.beta.1-4(Fuc
 .alpha.1-3)GlcNAc. As expected, only lanes 4-6 containing sCR1[des-A]sLex
 obtained from LEC-11 cells are positive for the appropriate carbohydrate
 structure.
 12. EXAMPLE 7
 12.1 MONOSACCHARIDE COMPOSITION OF SCR1[DES-A] IS CONSISTANT WITH SLE.sup.x
 GLYCOSYLATION
 The monosaccharide composition of the glycans comprising the carbohydrate
 moiety of the sCR1[des-A]sLex were analyzed using gas-liquid
 chromatography (GLC) following the procedure of Reinhold, V. (1972),
 Methods in Enzymology, 25:244-249. The conditions of cleavage,
 derivitization and GLC provided for a quantitative determination of the
 monocaccarides comprising the the glycoproteins sCR1[des-A] and
 sCR1[des-A]sLex.
 The CHO line utilized for the expression of sCR1 and sCR1[desLHR-A], CHO
 DUKX-B11, lacks .alpha.(1,3)fucosyl transferase activity and is thus
 incapable of sLex glycosylation. Stanley and colleagues have generated a
 mutant CHO cell line, LEC11, which transcribes endogenous alpha(1-3)
 fucosyltransferases and can synthesize carbohydrates with fucosylated
 terminal structures, including sLe.sup.x.
 In this example, LEC11 cells capable of sLe.sup.x glycosylation have been
 transfected with the plasmid encoding sCR1[desLHR-A] to produce
 sCR1[desA]sLe.sup.x. The sCR1[des-A]sLe.sup.x was compared to sCR1[des-A]
 produced in DUKX-B11 cells. When the two glycoproteins where analyzed for
 monosaccharide composition the results presented in Table I below were
 obtained:
 TABLE I
 sCR1[des-A] sCR1[des-A]sLex
 % Wt MR % Wt MR
 Fuc 1.4 1.1 2.7 1.9
 Man 4.1 3.0 4.7 3.0
 Gal 3.6 2.6 4.7 3.0
 GlcN 7.5 4.4 8.9 4.6
 NA 2.7 1.1 4.5 1.7
 The values presented in Table I for % Wt are the percentage of the
 particular sugar relative to the total protein. The MR is the molar ratio
 normalized to mannose. Fuc = fucose, Man = mannose, Gal = galactose, GlcN
 = N-acetyl-glucosamine, NA = sialic acid.
 It can be concluded from Table I that the fucose and sialic acid content of
 the sCR1[des-A]sLex are consistent with the expectation that the Lec 11
 cell line is adding the appropriate carbohydrates necessary for the
 sialylated Lewis x antigen as well as the sialylated di-Lewis x antigen.
 sLe.sup.x is the carbohydrate ligand for both P- and E-selectin and thereby
 mediates leukocyte adherence at vascular sites of inflammation.
 sCR1[desA]sLe.sup.x thus combines the anti-inflammatory potential of both
 a complement regulatory protein and an adhesion molecule.
 13. EXAMPLE 8
 13.1 MASS SPECTROSCOPY OF THE OLIGOSACCHARIDES FROM SCR1[DES-A]SLEX IS
 CONSISTANT WITH SLEX GLYCOSYLATION
 Mass spectroscopy confirmed the presence of fucose and sialic acid
 containing carbohydrates consistent with sLex glycosylation. Electron
 microspray (Fenn et al., (1989) Science, 246:64-71) followed by mass
 spectroscopy provided an assessment of the N-acetylneuraminic acid groups
 (sialic acid), fucosylation, and partial insight into antenna extensions
 and branching. In this Example sCR1[des-A]sLex was endo-H deglycosylated
 and an ES-MS "fingerprint" was obtained and compared to a similar
 "fingerprint" obtained from an endo-H deglycosylated sCR1[des-A]
 glycoprotein.
 Carbohydrates were grouped into bi- tri- and tetra antennary structures
 each having the typical trimannose core structure.
 In the resulting ion profile each ion was accounted for by reference to
 composition-mass tables compiled for each monosaccharide. The ion m/z
 1062.8 found in the sCR1[des-A]sLex "fingerprint", for instance,
 represents a biantennary structure consistant with a preferred sLex
 carbohydrate moiety and can be accounted for as fucosylated sialylated
 Lewis x antigen of the following structure:
 ##STR3##
 The corresponding structure with equivalent m/z is absent in the
 "fingerprint" analysis of sCR1[des-A].
 The data in Table II represents the percent mole ratio of each of the
 particular carbohydrate structures present in the sCR1[des-A] and
 sCR1[des-A]sLex compostions.
 TABLE II
 Glycoform* sCR1 sCR1[des-A] sCR1[des-A]sLex
 BiNA.sub.0 49.6 31.9 8.4
 BiNA.sub.0 F.sub.1 -- -- 3.6
 BiNA.sub.0 F.sub.2 -- -- 1.7
 BiNA.sub.1 19.6 27.0 15.8
 BiNA.sub.1 F.sub.1 -- -- 10.9
 BiNA.sub.1 F.sub.2 -- -- 4.6
 BiNA.sub.2 4.0 13.0 9.0
 BiNA.sub.2 F.sub.1 -- -- 9.6
 BiNA.sub.2 F.sub.2 -- -- 4.8
 TriNA.sub.0 7.4 5.4 1.6
 TriNA.sub.0 F.sub.1 -- -- &lt;1
 TriNA.sub.0 F.sub.2 -- -- &lt;1
 TriNA.sub.1 4.0 3.8 4.1
 TriNA.sub.1 F.sub.1 -- -- 1.9
 TriNA.sub.1 F.sub.2 -- -- &lt;1
 TriNA.sub.2 1.7 2.7 4.3
 TriNA.sub.2 F.sub.1 -- -- 3.5
 TriNA.sub.2 F.sub.2 -- -- &lt;1
 TriNA.sub.3 1.1 2.7 2.7
 TriNA.sub.3 F.sub.1 -- -- 2.0
 TriNA.sub.3 F.sub.2 -- -- &lt;1
 TetraNA.sub.0 1.8 1.3 --
 TetraNA.sub.1.sup.# 1.8 1.6 1.2
 TetraNA.sub.2.sup.# &lt;1 1.3 1.9
 TetraNA.sub.3.sup.# &lt;1 &lt;1 1.6
 TetraNA.sub.4.sup.# &lt;1 &lt;1 1.3
 BiNA.sub.0 -(Gal) 5.5 4.8 1.9
 BiNA.sub.1 -(Gal) &lt;1 1.8 &lt;1
 BiNA.sub.0 -(Fuc) 1.7 1.5 &lt;1
 *core fucosylated; approx. 3% are non-fucosylated
 #including sialyl lewis(x) in TP18
 ES-MS analisys of sCR1[desA]sLe.sup.x -derived oligossaccharide structures
 is consistent with sLe.sup.x glycosylation.
 14. EXAMPLE 9
 14.1 FUNCTIONAL ACTIVITY OF sCR1[des-A]sLex IN VITRO
 In this Example the functional activities of the purified proteins
 sCR1[des-A] and sCR1[des-A]sLex are compared in vitro. sCR1[desA]sLe.sup.x
 inhibited E-selectin mediated binding of U937 cells to activated human
 aortic endothelial cells in a concentration-dependent manner in vitro. In
 this static adhesion assay, sCR1[desA]sLe.sup.x inhibited binding of U937
 cells by 50% at a final concentration of 250 ug/ml.
 14.1.1 Static Adhesion Blocking Assay.
 Aortic endothelial cells (Clonetics), confluent in 96-well microtiter
 plates, were stimulated with TNF (100 U/ml) for 4 h at 37.degree. C. The
 cells were then washed twice with DNEN supplemented with 1% FBS. Serial
 dilutions of sCR1[desA]sLe.sup.x and sCR1[desLHR-A] were made to achieve
 final concentrations of 500, 250, 125, 62.5, and 0 ug/ml. To each well,
 5.times.10.sup.5 U937 (obtained from ATCC) cells in 20 .mu.l of DMEM were
 added and incubated for 20 min at 37.degree. C. The wells were filled with
 media, sealed, and centrifuged inverted at low speed (150.times.g) for 5
 min. The seal was removed, the plates blotted, and the number of bound
 cells in three microscope fields was determined.
 14.2 RESULTS
 Using this in vitro static adhesion assay, sCR1[desA]sLe.sup.x inhibited
 E-selectin mediated adhesion in a concentration-dependent manner. Human
 aortic endothelial cells were stimulated with TNF to induce cell surface
 expression of E-selectin. Surface expression of E-selectin was determined
 using DREG-56 (a monoclonal antibody specific for E-selectin) in an
 immunocytochemical staining protocol. U937 cells, shown to have surface
 sLe.sup.x by flow cytometric analysis with CSLEX-1, were shown to adhere
 to the activated endothelial cells. The adherence phenomenon between the
 activated aortic endothelial cells and U937 cells was shown to require the
 presence of calcium, a hallmark of selectin-mediated adhesion. The
 E-selectin dependent adhesion of U937 cells to activated endothelial cells
 was inhibited by sCR1[desA]sLE.sup.x in a concentration dependent manner.
 FIG. 3 details the results of this experiment. The black bars represent the
 sCR1[des-A] material obtained from DUKX-B11 cells. The bars with
 horizontal lines represent sCR1[des-A]sLex material obtained form LEC-11
 cells. The sCR1[des-A]sLex material inhibited binding of U937 cells to
 activated aortic endothelial cells in a concentration dependent manner.
 15. EXAMPLE 10
 15.1 IN VIVO FUNCTIONAL ACTIVITY OF sCR1[des-A]sLex
 Endothelial upregulation of selecting, to which oligosaccharides such as
 sialyl Lewis.sup.x, and sialyl di-Lex bind, are important adhesion
 promoting molecules for neutrophils. The soluble complement receptor 1
 (sCR1), which is a potent inhibitor of complement, has been expressed in a
 truncated form, with and without decoration with SLe.sup.x
 (sCR1[desA]sLe.sup.x and sCR1[desA], respectively). Both compounds have
 substantial complement-blocking activity in vitro as demonstrated above.
 In a rat model of P-selectin-dependent acute lung injury, the rank order
 of protective activity for these inhibitors is: sCR1[desA]sLe.sup.x
 &gt;sCR1.gtoreq.sCR1[desA]. By taking advantage of oligosaccharide decoration
 of sCR1[desA] to cause binding to the activated endothelium at sites of
 selectin expression, the complement inhibitor can be "targeted" to an
 inflammatory site.
 The inhibitor preparations were employed in vivo in the CVF model of rat
 lung injury. Four separate groups of rats (n=5 each) were pretreated
 intravenously with 0.4 ml sterile saline, sCR1, sCR1[desA] or
 sCR1[desA]sLe.sup.x (each at 15 mg/kg body weight) and injected
 intravenously 5 min before intravenous infusion of CVF. Also, a negative
 control group (infused with sterile saline in the absence of CVF) was
 employed (n=5). Thirty min. after infusion of CVF or sterile saline,
 animals were killed with an overdose of ketamine and 1.0 ml blood obtained
 from the inferior vena cava (a).
 As shown by the data in FIG. 4, treatment of animals with sCR1[desA],
 sCR1[desA]sLe.sup.x or sCR1 reduced (as a percentage) MPO content in lung
 by 40.+-.3, 64.+-.3 and 55.+-.4, respectively (FIG. 4C). FIG. 4C is a
 measure of the accumulation of neutrophils in the lung as estimated by
 measurement of myeloperoxidase activity (MPO). When compared
 statistically, sCR1[desA]sLe.sup.x and sCR1 were more effective than
 sCR1[desA] in reducing MPO content.
 FIG. 4B also describes the protective effects of sCR1, sCR1[des-A], and
 sCR1[des-A]sLex from hemorrhagic lung injury induced by CVF. FIG. 4B is
 the measurement of the reduction over control of hemorrhage as measured by
 a radiolabelled red blood cell leakage into the lung from the blood
 vessel. sCR1[des-A]sLex reduced hemorrhage approximately 65 percent over
 control. Permeability is a measure of radiolabelled protein leakage from
 the blood vessels of the lung. sCR1[des-A] reduced permeablity
 approximately 60 5 over control in this experiment. Thus,
 sCR1[desA]sLe.sup.x appears to be the most effective of the three
 complement inhibitors in reducing injury in this inflammatory model.
 At the time of sacrifice (30 min after intravenous infusion of CVF), plasma
 was obtained and evaluated for the concentration of sCR1[desA] antigen
 using an ELISA sandwich technique. Plasma from CVF infused animals that
 were otherwise untreated revealed &lt;50 ng/ml measurable sCR1[desA] antigen,
 while plasma antigenic levels of sCR1[desA] in the sCR1[desA] and
 sCR1[desA]sLe.sup.x treated animals (injected with CVF) were 267.+-.28.2
 and 154.+-.33.9 .mu.g/ml, respectively. These data would be consistent
 with an accelerated selectin-dependent removal of sCR1[desA]sLe.sup.x from
 the vascular compartment.
 These data demonstrate that, in the P-selectin-dependent model of acute
 lung injury occurring after CVF-induced systemic activation of complement,
 the complement inhibitor, sCR1[desA] decorated with sLex groups provides
 the most effective protection (as compared to sCR1[desA] or sCR1) in this
 model of neutrophil-dependent injury. Reduced MPO content in lung suggests
 that sCR1[desA]sLe.sup.x more effectively blocked P-selectin-dependent
 adhesion of neutrophils to the activated endothelium, which is known to be
 upregulated for P-selectin. Each of three complement inhibitors had
 protective effects that were associated with diminished buildup of lung
 MPO.
 By reducing the amount of endothelial activation (upregulation of
 P-selectin) and diminishing neutrophil activation (resulting in generation
 of toxic oxygen products), complement blockage interferes with
 injury-promoting interactions between neutrophils and the endothelium. In
 this model of lung injury it is known that both neutrophils and toxic
 oxygen products are required for full development of injury. The close
 proximity between neutrophils and the endothelium is required for the most
 effective action of toxic oxygen products (from neutrophils) on the
 endothelium. These adhesive interactions can be blocked with antibodies to
 P-selectin or leukocytic .beta.2 integrins, ox by infusion of sLe.sup.x.
 In all cases the protective effects of these interventions are associated
 with diminished levels of tissue MPO. The enhanced inhibitory activity of
 sCR1[desA]sLe.sup.x would be consistent with the interpretation that, as
 CVF-induced complement activation occurs (thus causing endothelial
 upregulation of P-selectin), sCR1[desA]sLe.sup.x can selectively bind to
 endothelial P-selectin, providing localized protection against further
 complement activation. Localization of sCR1[desA]sLe.sup.x to areas of
 activated endothelium is supported by the immunostaining data and could
 also explain why residual plasma levels of sCR1[desA] antigen at 30 min
 were nearly 50% lower in sCR1[desA]sLe.sup.x treated animals than those
 treated with sCR1[desA].
 The ability to "target" complement inhibitors to the endothelium based on
 the ability of sLex to cause binding of sCR1[desA]sLe.sup.x to P-selectin
 (or to E-selectin) provides a unique strategy to optimize the protective
 effects of these inhibitors. Since ischemia-reperfusion injury to the
 myocardium appears to be P-selectin-dependent, it is possible that in
 humans treatment of ischemia-reperfusion injury would benefit from the use
 of such inhibitors, as well as other conditions in which selectin and
 complement activation molecules participate in outcomes leading to injury.
 16. EXAMPLE 11
 16.1 GENERATION OF A SOLUBLE COMPLEMENT RECEPTOR TYPE 1 WITH SELECTIN
 BINDING ACTIVITY
 We describe herein another soluble form of complement receptor type 1 with
 selectin binding activity. This bifunctional molecule is a valuable tool
 in modulating the inflammatory response.
 16.1.1. CELL LINES
 The cell line K562 was supplied by Dr. Lloyd Klickstein, Center for Blood
 Research, 200 Longwood Avenue, Boston, Mass. 02115, and is generally
 available for the American Type Culture collection (Rockville, Md.). HL-60
 cells were obtained from the ATCC.
 16.1.2. MONOCLONAL ANTIBODIES
 Rabbit polyclonal antiserum specific for CR1 can be obtained by standard
 techniques known in the art by immunizing rabbits with human complement
 receptor type 1. Monoclonal antibodies to CD15 are commercially available
 and can be obtained from Dako, California and for instance, clone 28 may
 be obtained from AMAC, Inc., Maine. Murine monoclonal antibody 3C6. D1l
 was obtained from a standard fusion using the method originally described
 by Kohler and Milstein (1975, Nature 256:495-497). Balb/c mice were
 immunized at 3-4 week intervals with purified recombinant complement
 receptor type 1 i.p. in Freunds adjuvant. Four weeks after the third
 immunization, mice were boosted intravenously with 10 .mu.g CR1 and the
 spleen was removed four days later. Spleenocytes were fused with NSO
 myeloma cells by addition of 1 ml of 50% PEG-1500 (Boehringer Mannheim,
 Indianapolis Ind.), then diluted with 20 ml of OPTI-MEM media (GIBCO).
 After fusion, the cells were plated into wells of 96 well flat bottomed
 plates and selected in medium containing HAT (GIBCO). Wells positive for
 growth were screened for the production of anti CR1 mAbs using a CR1
 capture antibody. Control antibodies were murine IgM and murine IGg1,
 commercially available from Becton Dickinson, Franklin Lakes, N.J., and
 Tago, Calif.
 16.1.3. TRANSFECTION
 K562 cells expressing complement receptor type 1 (CR1) can be obtained by
 transfecting host cells by electroporation with full length CR1 obtained
 from construct piABCD (Klickstein, et al., 1988, 168:1699). Approximately
 five million K562 cells suspended in 0.8 ml medium are mixed with
 approximately 20 .mu.g plasmid DNA, linearized with SpeJ, and subjected to
 200 volts, 960 .mu.F using a genepulser electroporation apparatus
 (BioRad). After several days in culture cells expressing the soluble CR1
 gene product can be selected for the expression of soluble CR1 using the
 CELLFREE.RTM. CD35 Bead Assay Kit obtained from T Cell Diagnostics, Inc.
 Cambridge, Mass.
 Alternatively, the calcium phosphate-mediated transfection of K562 cells
 can be accomplished using the method of Graham and van der Erb (1973)
 Virology 52:456-467.
 16.1.4. CELL LYSATES
 Cell lines transfected to express the appropriate molecules or cell line
 endogenously expressing the appropriate molecules were solubilized at
 5.times.107 cells/ml in lysis buffer containing 10 mM Tris pH 8.0, 1%
 nonidet P-40 (NP-40), 10 mM iodoacetamide (IAA), 1 mM phenylmethly
 sulfonyl fluoride (PMSF), 0.04% aprotinin and 0.3 mM
 N-tosyl-L-phenylalanine chloromethyl ketone (TPCK).
 16.1.5. WESTERN BLOT ANALYSIS
 The reactivity of CR1 purified by affinity chromatography from K562
 supernatants was tested by Western blot analysis. Supernatants from K562
 transfected with the full length CR1 were fractionated by 4-20% SDS-PAGE
 and then transferred to nitrocellulose sheets. The sheets were first
 blocked with blocking buffer (1% bovine serum albumin in
 phosphate-buffered saline in PBS). After blocking, the sheets were
 incubated with either antibody 3C6. D11 about 2-3 .mu.g/ml(anti-CR1),
 anti-CD15 (about 20 .mu.g/ml, or irrelevant isotype matched control
 antibody C305 (IgM, about 20 .mu.g/ml), or W112 (IgG, about 2-5 .mu.g/ml).
 After 1-2 hour incubation in the presence of the primary antibodies the
 sheets were washed with a solution of PBS and 0.05% Tween-20. After
 washing the sheets were incubated with horseradish peroxidase (HRP)
 conjugated goat anti-mouse antibody. After washing, color was developed
 with an HRP substrate,
 16.2. RESULTS
 As expected, the material recovered from the K562 cell culture supernatants
 can be detected by Western blot using antibodies to the Lewis X antigen as
 well as monoclonal anti-CR1 antibodies.
 16.3. PHYSICAL CHARACTERIZATION OF KCR1
 To define the specific carbohydrate structures of the KCR1 recovered supra
 both affinity purified KCR1 and neuraminidase treated KCR1 were tested for
 their ability to bind anti-CR1 immobilized on wells of 96 well plates.
 Detection was with an anti-CD15 antibody which is reactive with the Lex
 SLe.sup.x ligand structures. Treatment of the KCR1 with neuraminidase
 removes terminal sialic acid residues from the SLe.sup.x oligosaccharide
 structures yielding the Lewis X structure. Results of this analysis are
 presented in Table III.
 TABLE III
 Reactivity of KCR1 with anti-CD15 Antibody
 Test Sample Bound (OD.sub.490-650) Monoclonal Antibody
 CR1.sup.1 0.059 .+-. 0.002
 KCR1.sup.2 0.135 .+-. 0.001
 nKCR1.sup.3 0.130 .+-. 0.006
 KCR1.sup.4 0.110 .+-. 0.024
 All samples are the mean plus or minus the standard deviation of the mean
 for four samples excluding the control which is the average of two
 samples.

FNT .sup.1 CR1 represents a sample of CR1 obtained from Chinese Hamster ovary
 cells which does not contain appropriate carbohydrate structures for
 binding the CD15 antibody.

FNT .sup.2 KCR1 represents a sample of affinity purified CR1 produced in K562
 cells. The sample was concentrated by a Centricon.TM. concentrator prior
 to assay

FNT .sup.3 nKCR1 represents a sample of CR1 produced in K562 cells which was
 treated with neuraminidase. Neuraminidase treatment consisted of
 incubating the sample in the presence of neuraminidase prior to assay.

FNT .sup.4 KCR1 represents a sample of CR1 affinity purified from K562 cells
 and untreated prior to assay.
 17. GENERALIZED ASSAY FORMATS TO DETECT FUNCTIONAL ACTIVITY
 The compositions of the invention may also be evaluated for their ability
 to block intercellular adhesion to certain cells, for instance, activated
 endothelial cells thereby inhibiting a primary event in the inflammatory
 response. This evaluation may be achieved by a number of methods; the
 following methods being described as specific procedures that were
 employed in this regard or that may be useful in addition thereto:
 A. COMPETITIVE INHIBITION OF HL-60 BINDING TO E AND P SELECTINS
 a) cells expressing E or P selectin(activated platelets or cells
 transfected with and expressing selectins on their surface, ref Larsen,et
 al.) are grown in 96-well microtiter plates to confluence.
 b) HL-60 cells are added at 4 deg Centigrade in the presence or absence of
 CR1 or CR1 analogues and allow to settle and bind for 30 min.
 c) non-adherent cells are removed by inverting the plates and centrifuging
 at 150 xg for 5 min.
 d) the plates are scored for the number of bound HL-60 cells per microscope
 field.
 B. IN VIVO ASSAY FOR SELECTIN BINDING
 a) induce P-selectin up-regulation in rats with CVF in accordance with the
 method of Mulligan et al., 1992, J. Clin. Invest. 90: 1600-1607 .
 b) inject radio-labelled sLEX-CR1 chimera or analogues vs TP10 and
 determine distribution of radiolabel.
 C. IN VIVO ASSAY FOR COMPOSITION EFFICACY
 Mulligan et al.("Role of Leukocyte Adhesion Molecules in Complement-Induced
 Lung Injury", J. Immunol. Vol. 150, 2401-24061, No. 6, Mar. 15, 1993,)
 describe the role of P selectin in lung vascular endothium injury in rats
 after cobra venom factor (CVF) activation of complement. Since it has
 previously been shown that complement has a protective effect in
 preventing acute microvascular injury of the lung induced by CVF, it is
 desirable to home the CR1 to the site of the injury via the selectin
 ligand. In order to assess the localization of the complement, twenty
 units of CVF per kg body weight is injected intravenously into male
 300-350 gram Long Evans rats. To assess the localization of the CR1 to the
 lung .sup.125 I-CR1-sLe.sup.x (approximately 500 .mu.Ci), or in control
 animals .sup.125 I-CR1 is injected at for instance 15 mg/kg body weight.
 Since CVF induced lung injury is instantaneous, localization can be
 assessed by assessing tissue incorporation of radiolabelled CR1-sLe.sup.x
 by standard techniques approximately 30 minutes after injection.
 The CVF model can also be used to assess the ability of the sLexto prevent
 the primary events in inflammation such as neutrophil sequestration and
 subsequent rolling and firm attachment. See also, Mulligan et. al., Role
 Endothelial-Leukocyte Adhesion Molecule 1 (ELAM-1) in Neutropnil-Mediated
 Lung Injury in Rats, J. Clin. Invest., Vol. 88, October 1991, 1396-1406,
 and Mulligan et al., "Neutrophil-dependent Acute Lung Injury," J. Clin.
 Invest., Vol. 90, October 1992, 1600-1607.
 18. OTHER TECHNIQUES FOR PREATIONS OF COMPOSITIONS
 18.1. MUTAGENESIS
 CHO (Chinese hamster ovarian) cells that express sCR1 are used. The cells
 in suspension (at about 2.times.10.sup.5 cells/ml) can be incubated for 18
 hrs with EMS, washed, and relative plating efficiencies determined.
 Mutagenized cells may be cultured for seven days to allow expression of
 acquired mutations. Cells can be aliquoted at about 10.sup.6 cells/100-mm
 tissue culture dish in medium containing about 10% fetal calf serum and
 the appropriate concentration of the primary selective lectin(s). After
 six days, the plates are washed twice with alpha medium and the scondary
 selective lectin(s) added in alpha medium containing 10% fetal calf serum.
 After approximately four more days of incubation, the largest colonies are
 picked into alpha medium containing 10% fetal calf serum and the plates
 stained with 2% methylene blue in 50% methanol. Control plates which
 contained no lectin or only the primary selective lectin(s) are stained
 after 8 days and relative plating efficiencies determined.
 18.2. CELL FUSION
 Approximately 1.times.10.sup.8 cells expressing the .alpha.1,3-fucosyl
 transferase and 5.times.10.sup.7 cells expressing the desired complement
 protein which have been previously suspended in DMEM, were pelleted by
 centrifugation at 200.times.g for 5 minutes and warmed to 37.degree. C.
 The pellet was resuspended in 1 ml of culture medium containing about 1
 g/ml of polyethylene glycol (PEG) 4000, supplemented with 5% DMSO at
 37.degree. C. with gentle mixing. The cells wee then spun at 100.times.g
 for 2 minutes, 4.5 ml of supplemented medium was added over the next 3
 minutes followed by 5 ml of supplemented medium over the next two minutes.
 Then the tube was filled with supplemented medium. As is well known in the
 art, timing of these steps is important.
 The cells were pelleted by centrifugation at 100.times.g for 5 min at room
 temperature, then the supernatant was aspirated. The cell pellet was
 resuspended in medium, but care was taken not to force the dispersion of
 small cell clumps. The cells were plated in a 96-well plate in limiting
 dilution (To ensure growth, the wells of the plate may contain feeder
 cells). Culture medium containing mycophenolic acid was added, and then
 replaced as often was deemed necessary to ensure cell selection. Cells
 expressing sLex or sialyl di-Lex are then selected.
 The present invention is not to be limited in scope by the specific
 embodiments described herein. Indeed, various modifications of the
 invention in addition to those described herein will become apparent to
 those skilled in the art from the foregoing description and the
 accompanying figures. Such modifications are intended to fall within the
 scope of the appended claims.
 Various publications are cited herein, the disclosures of which are
 incorporated by reference in their entireties.