LECTIN DOMAIN VARIANTS OF SELECTIN

Abstract: This invention concerns selectin variants having an amino acid alteration at a site or sites of a selectin lectin domain amino acid sequence within a region defined by binding sites recognized by blocking monoclonal antibodies to the corresponding selectin unaltered in the lectin domain. Nucleotide sequences encoding such variants, expression vectors containing such nucleotide sequences, and host cells transformed with such expression vectors are also within the scope of the invention.

Description: LECTIN DOMAIN VARIANTS OF SELECTIN . BACKGROUND OF THE INVENTION I. Field of the Invention This invention relates to selectin variants. The invention further relates to nucleic acids encoding, and to methods and means for preparing these variants. II. Description of Background and Related Art The selectins are cell adhesion molecules that are unified structurally by the inclusion of lectin, egf-like and complement binding¬ like domains [Bevilacqua, M.P., et al . , Science 243. 1160-1165 (1989); Johnson, et al., Cell 56, 1033-144 (1989) ; Lasky, L.A., Cell 56. 1045- 1055 (1989); Siegel an, M. et al . , Science 243. 1165-1172 (1989); Stoolman, L. . , Cell 56: 907-910 (1989)] , and functionally by their ability to mediate cell binding through interactions between their lectin domains and cell surface carbohydrate ligands [Brandley, B., et al. Cell 6.3, 861-863 (1990); Springer, T. , and Lasky, L. A. Nature 349 196-197 (1991)] . There are three members identified so far in-the selectin family of cell adhesion molecules: L-Selectin (a.k.a. peripheral lymph node homing receptor (pnHR) , LEC-CAM-1, LAM-1, gp90 MEL , gpl00 MEL , gpll0 MEL , MEL-14 antigen, Leu-8 antigen, TQ-1 antigen, DREG antigen) , E-Selectin (LEC-CAM- 2, LECAM-2, ELAM-1) and P-Selectin (LEC-CAM-3, LECA -3, GMP-140, PADGE ) . The structures of the selectin family members are illustrated in Figure 9. L-Selectin is found on leukocytes and is involved with the trafficking of lymphocytes to peripheral lymphoid tissues [Gallatin et al., Nature 303. 30-34 (1983)] and with acute neutrophil-mediated inflammatory responses [Watson, S.R., Nature 349 164-167 (1991)] . The amino acid sequence of L-Selectin and the encoding nucleic acid sequence are, for example, disclosed in ϋ. S. Patent No. 5,098,833 issued 24 March 1992. L-Selectin appears to recognize sialylated, fucosylated, sulfate carbohydrate ligand(ε) on at least two endothelial glycoproteins [True, D.D., et al., J .Cell Biol. Ill, 2757-2764 (1990) ; Imai, Y. et al., Cell Biol. 113 1213-1221 (1991)] , one of which has recently been clone [Lasky, L. A. et al., Cell (1992), in press and copending U.S. Application Serial No. 07/834,902 filed 13 February 1992] . E-Selectin is an endothelial adhesion molecule that is induced b various inflammatory stimuli [Bevilacqua, P. P. et al., Proc. Natl. Acad. Sci. USA 84.. 9238-9242 (1987) ; Luscinskas, F.W. et al . , J. Immunol. 14 2257-2263 (1989); Kuijpers, T.W. et al . , . Immunol . 147 1369-137 (1991)] . A cloned gene encoding E-Selectin (ELAM-1) is disclosed in U. S. Patent No. 5,081,034 issued 14 January 1992. E-Selectin recognize the neutrophil and monocyte cell surface carbohydrate, sialyl Lewis x (sLex) [Lowe J. B. et al.- Cell 63 475-484 (1990) ; Phillips, M. L. et al.. Science 250 1130-1132 (1990); Walz, G.A- et al. Science 250 1132- 1135 (1990) ; Tiemeyer, M. et al., Proc. Natl. Acad. Sci- USA 88 1138-1142 (1991) ] r and in addition, may also be involved with the recognition of an sLex-like carbohydrate on the surface of a skin-homing subset of lymphocytes [Picker, L.J. et al., Nature 349 796-799 (1991); Shimizu, ϊ. et al., Nature 349. 799-802 (1991)] . The minimum sized sLex-related carbohydrate recognized by E-Selectin is a tetrasaccharide of the structure Sialic Acid alpha2-3 Galactose beta 1-4 N-Acetyl Glucosamine(Fucose alpha 1-3) [Tyrrell, D. et al., Proc. Natl. Acad. Sci. USA. 88. 10372-10376 (1991)] . P-Selectin is found in alpha granules of platelets and Weible-Palade bodies of endothelial cells [Bonfanti, R. et al. , Blood 73. 1109-1112 (1989); McEver, R. et al., J- Clin- Inv. 84, 92-99 (1989)] - Its surface ea ression is induced within minutes of exposure to thrombin, substance P, histamine or peroxide, and it appears to recognize a carbohydrate that is either identical to or closely related to sLex on both neutrophil and monocyte cell surfaces [Larsen, E. et al., Cell 59. 305-312 (1989); Larsen, E. et al. Cell 63, 467-474 (1990); Moore, K.L. et al . J. Cell Biol. 112. 491-499 (1991); Polley, M. J. et al., Proc. Natl. Acad. Sci. USA 88. 6224-6228 (1991)] . The P-Selectin amino acid and the encoding nucleotide sequences are disclosed by Johnston et al., Cell 56, 1033-1044 (1989) . The lectin domains of L-, E- and P-Selectins show remarkable sequence homology and structural similarity. Particularly noteworthy is the conservation of cysteine (Cys) residues at amino acid positions 19, 90, 109 -and 117 of the selectin lectin domains, which results in a three dimensional structure comprising two-disulfide bonded loops defined by disulfide bonds formed between Cysl9 and Cysll7, and Cys90 and Cysl09, respectively. Much evidence has accumulated to indicate similarities in the nature of the carbohydrate ligands seen by selectins. In the case of all three selectins, the adhesive interactions between their lectin domains and carbohydrate ligands require the presence of alpha 2-3 linked sialic acid fucose residues [Brandley, B. e al.. Cell 63. 861-863 (1990); Corrall, . e al., Biochem- Biphvs. Res. Commun. 172 , 1349-1352 (1990); Springer, T. and Lasky, L. A. Nature 9 196-197 (1991) ; Tyrrell, D. et al., Proc. Natl. Acad- Sci. USA 88. 10372-10376 (1991)] . The adhesive interactions between selectin lectin domains and their carbohydrate ligands may be relatively weak, since L- and P-Selectin have been shown to be involved in the - relatively low affinity "rolling" of leukocytes along the endothelium during inflammatory responses [Lawrence, M. B. ec al., Cell 65., 859-873 (1991); Ley, K. e al. Blood 77(12) , 2553-2555 (1991) ; Von Andrian, V. e l., Proc. Natl. Acad. Sci. USA 88, 1538-1542 (1991)] . The molecular details of the interactions between selectin lectin domains and their carbohydrate ligands are poorly understood. The fact that all three selectins require sialic acid for adhesion, when coupled with the finding that certain negatively charged carbohydrate polymers, such as. fucoidin, dextran εulfate, and polyphoεphomannan ester, are effective inhibitors of some selectin-mediated cell adhesion, is consistent with the involvement of positively-charged amino acids in carbohydrate recognition. However, that such protein-sialic acid interactions can also be accomplished by non-charged side chains is suggested by crystallographic analysis of the low-affinity interaction between the influenza hemagglutinin glycoprotein, which is not related to type C lectinε, and its cell surface ligand, sialic acid, which has revealed that this interaction involves a diversity of amino acid side chains, none of which are positively charged [Weis, W. et al., Science 254, 1608-1615 (1985)]. That a simple face or pocket of the E-, and potentially P- [Polley, M. J. et al., Proc. Natl. Acad. Sci. USA 88, 6224-6228 (1991)] Selectin lectin domain is involved with the recognition of sLex is suggested by NMR solution analyses of the sLex structure which demonstrate that the critical sialic acid and fucose residues both point to one face of thiε carbohydrate ligand and are εeparated by -10 angstromε, while an inactive form of thiε carbohydrate (with a 2-6 linked sialic acid) has these two important functional components pointing in very different directions. A similar structural analysis of another ligand for E-Selectin, sialyl Lewis a (sLea: Sialic Acid alpha 2-3 Galactose beta 1-3 N-Acetyl Glucosamine(Fucose alpha 1-4)), has revealed that the critical sialic acid and fucose residues again point to one face of the tetrasaccharide and are separated by approximately the same distance as they are in sLex. See Berg, E.L. et al . J . Biol. Chem. 265, 14869-72 (1991); Tyrrell, D. et al. (1991), Supra. An object of the present invention is to identify the region(s) within the amino acid sequence of selectin lectin domains that is/are critical for the interaction of selectin receptors and their ligands. It. is another object to enable the preparation of amino aci sequence variants of selectins having improved ligand binding properties, in particular increased affinity for their respective ligands as compare to the corresponding native selectin receptors. A further object is to identify selectin lectin domain sequence that are not critical for the interaction of selectins and thei respective ligands. It. is another object to provide selectin amino acid sequenc variants with improved pharmacological characteristics, e. g. increase physical and/or chemical stability, increased half-life, decreased rat of in vivo clearance, decreased severity or occurrence of side effect during therapeutic use, etc., having retained or increased ligand bindin affinity as compared to the corresponding native selectin. Selectin variants with enhanced ligand binding properties have great therapeutic potential as effective inhibitors of pathologic inflammatory responses mediated by selectins by blocking the εelectin-selectin ligand interaction. These and further objects of the present invention will be apparent for one skilled in the art. SUMMARY OF THE INVENTION The adhesive interactions between sLex and selectin lectin domains were analyzed following two strategies. In the first, chimeric human- rabbit selectin lectin domains were generated based upon amino acid sequence differences between the two species. These chimeras have then been used to map epitopes recognized by anti-human selectin blocking monoclonal antibodies (Mabs) . In the second, selectin point mutants were generated and analyzed for binding to a panel of blocking and non- blocking -anti-selectin Mabs and for their ability to adhere to immobilized sLex glycolipid. Residues affecting various aspectε of selectin structure and/or function have then been superimposed onto a three-dimensional model of the selectin lectin dσmainε that has been generated using the structural coordinates determined for a related C type lectin, the mannose binding protein. [Weiss, ff. et al . , Science 254 1608-1615 (1991)] . Together, these data define a relatively small region within the selectin lectin domain that is critical for recognition of the carbohydrate ligand, sLex. It has been found that the selectin amino acid residues primarily involved with carbohydrate recognition are within a patch on the surface of the lectin domain, near the antiparallel beta sheet formed by the disulfide-linked N- and C-termini and the conformationally adjacent disul ide loop formed by the two internal cysteines. Accordingly, εLex is apparently recognized by a relatively small region of the selectin lectin domain, comprised of residues from the N- and C-termini and the small disulfide-bonded loop. It has further been found that positively-charged amino acid side chains have an essential role in the recognition of carbohydrate ligands of selectins. It has additionally been, found that the replacement of charged amino acid residues by uncharged residues in the N-terminal region of selectinε increaεe the binding affinity of selectinε to their respective carbohydrate ligands. In one aspect, the present invention provides a selectin amino acid sequence variant having an amino acid alteration at a site or sites of the selectin lectin domain recognized by blocking monoclonal antibodies to the corresponding unaltered selectin- In a preferred embodiment, the amino acid alteration is at a site or sites within a patch comprising amino acids on the surface of the lectin domain near the antiparallel beta strand formed by the disulfide- linked N- and C-termini of the lectin domain and the amino acids of the adjacent disulfide loop formed by the two internal cyεteines of the lectin domain. In one particularly preferred embodiment, the amino acid alteration is within the patch defined by amino acid residue numbers 1-9, and 90 to the C-terminus of the lectin domain of the corresponding-native εelectin. In a further preferred embodiment, the amino acid alteration is within the three dimensional patch defined by amino acid residue numbers 7-9, 90-109 and 113 of the lectin domain of the corresponding native selectin. In an even more preferred embodiment, the alteration is at one or more of amino acid positions 7-9, 43-48, 82-86, 94-100 and 113 of the coresponding native human selectin. In a still further preferred embodiment, the amino acid alteration is at a irio acid residue numbers 7-9, or 84-86, and most preferably at amino acid residue number 8 or at amino acid residues 84 and 86. In all cases, the amino acid alteration preferably is substitution. In a particularly preferred embodiment, the amino acid alteration is the substitution of an small uncharged amino acid for a charged amino acid at amino acid residue number 8 of the lectin domain of the corresponding native selectin. The variants preferably have a positively charged amino acid at at least one of the amino acid positions 97, 111 and 113 of the lectin domain of the corresponding native selectin. In a still further preferred embodiments, the selectin amino acid sequence variants herein retain the egf like domain of a corresponding selectin. In other embodiments, this invention relates to a nucleic acid sequence encoding the selectin variant described above, replicable expreεεion vectorε compriεing and capable of expressing,the nucleic acid sequence in a transformant host cell, and microorganisms and cell cultures transformed with the vector. In a still further embodiment, the invention provides a metho comprising: (a) introducing an amino acid alteration into a selectin lecti domain at a site or sites recognized by blocking monoclonal antibodie to the corresponding unaltered selectin; and (b) screening the resultant selectin variant for enhanced bindin affinity to a correεponding εelectin ligand. The selectin variants of the preεent invention with enhanced bindin affinity for their reεpective native ligands can be used to block th binding of a corresponding native selectin to its ligand, an accordingly, are useful as inhibitors of pathologic inflammator responses mediated by selectins. For example, such L-Selectin variants (L-Selectin agonistε) effectively block the binding of the L-Selectin on a circulating leukocyte to its native ligand on an endothelial cell. This property is useful for treating a symptom or condition associated with excessive binding of circulating leukocytes to endothelial cells, such as inflammation associated with rheumatoid arthritis, psoriasis, multiple sclerosis, etc. Accordingly, the present invention also provides a method for the treatment or prevention of a symptom or condition associated with a pathologic inflammatory response mediated by a selectin comprising administering to a patient having or at risk to develop such symptom or condition a therapeutically effective amount of a selectin amino acid εequence variant having enhanced ligand binding affinity. The selectin variants of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the variant is combined in admixture with a pharmaceutically acceptable carrier- Such pharmaceutical compositions are within the scope of the present invention. The present invention further concerns chimeric proteins comprising a fusion of an amino acid εequence comprising a selectin lectin domain sequence with an alteration as hereinabove described (preferably along with egf like domain and short concensus repeat (SCR) sequences) to an immunoglobulin constant domain sequence- In a further aspect, the present invention provides a hybrid selectin comprising a portion of a selectin from an animal species with complementary part or parts or a different type of εelectin of the same animal species or of the same type of εelectin of a different animal εpecies- In a still further aspect, this invention relates to an anti-E- Selectin monoclonal antibody composition capable of binding to substantially the same E-Selectin lectin domain epitope recognized by or which compete with, a monoclonal antibody selected from the group consisting of 7H5; 8E4; 3B7; 1D6; 4D9; 1E5; 9A1; 7E10; 1B3; 1 G2; 11G5; and 9H9. Selectin variants having reduced affinity for their native ligand are useful in screening assays for substances (e.g., peptides, fermentation broth components, carbohydrate derivatives and the like) capable of blocking or enhancing the binding of the εelectin with its ligand. In one embodiment, the ability of the substance to be tested to bind to. the variant selectin is determined. If it is εubεtantially unable to bind to the variant εelectin and to the ligand, but does affect the binding of native selectin to its ligand, then one may conclude that it interacts with the selectin at residueε instrumental in ligand binding- Such substances are of particular interest as antagonists of selectin-ligand binding- In another embodiment, the selectin variants are used to immunize animals so as to identify antibodies that bind to other domains of the selectin. In an additional embodiment, the variants are used to screen for other antibodies that bind specifically to the ligand binding domain of the selectin. In this embodiment, antibodies raised against the native selectin are screened for their ability to bind to the- selectin variant of thiε invention. Antibodies that subεtantially do not bind to a variant of this invention are selected as being capable of binding to the ligand binding site. Other diagnostic embodiments will be apparent to the ordinary artisan. BRIEF DESCRIPTION OF THE DRAWINGS Figure l. Characterization of Anti-E-Selectin Monoclonal Antibodies. (A) Inhibition of HL60 cell adhesion to cytokine activated HUVECs (solid bars) and E-Selectin transfected COS cells (striped bars) by anti-E-Selectin monoclonal antibodies. Cells were preincubated with MAb's (10 ug/ml) for 1 hr at room temperature and HL60 cell adhesion determined as described in the Experimental Procedures section of Example 1. Each bar represents the mean +/- SD of triplicate determinations expressed as a percent of control binding. (B) Binding of anti-E-Selectin MAb's to human (solid bars) and rabbit (striped bars) E-Selectin. Antibodies (10 μg/ml) were incubated for 1 hr at room temperature on 96 well microtitre plates to which recombinant soluble human or rabbit E- Selectin had been bound. Wells were washed and incubated with horseradish peroxidase coupled goat anti-mouεe IgG, washed and developed usin standard protocols. Figure 2. E-Selectin Constructε. (A) Amino acid alignment of the lectin domainε of human and rabbit E-Selectin beginning at the putative N-terminus of the mature polypeptide. Amino acid substitutions in the rabbit peptide are depicted, all other positions are identical with th human residues. (B) E-Selectin constructs used to map Mabs and sLe binding εites. The human E-Selectin contained amino acids 1-157, th rabbit E-Selectin encoded amino acids 1-156 and HuRa-l containe residueε 1-9 of human contiguouε with residues 10-156 of rabbit E- Selectin. Each is anchored to the cell surface via a GPI link encode by CD16 sequences. Also shown is an E-Selectin-IgG chimera (E Sel-IgG) containing the lectin, egf-like and complement binding-like domains (CBD) 1 and 2 of human E-Selectin ligated to the hinge, CH2 and CH3 regions o human IgG 1 [Watson et al . . , J. Cell Biol. 115. 235-243 (1990)] . Figure 3. Binding of blocking antibodies to HuRa-l chimera Immunofluorescence localization of anti-E-Selectin antibody binding t human E-Selectin lec-egf-CD16 (column 1) , rabbit E-Selectin lec-egf CD1 (column 2) and HuRa-l, human-rabbit chimeric E-Selectin lectin-egf CD1 (column- 3) transfected COS cells. Transfected cells were fixed an stained with anti-E-Selectin Mabs 3B7 (row 1), 7H5 (row 2), 8E4 (row 3) or 14G2 (row 4) . Figure 4. HL60 binding to HuRa-l in the presence of blocking antibodies. Inhibition of HL60 binding to human E-Selectin lec-egf-CD16 (solid bars) , rabbit E-Selectin lec-egf-CD16 (open bars) , and HuRa-l human-rabbit chimeric E-Selectin lectin-egf-CD16 (striped barε) tranεfected COS cells by anti-E-Selectin blocking Mabs. Cells were preincubated with 3B7, 7H5, 8E4, or 14G2 (10 μg/ml) for 1 hr and HL60 cell adhesion determined as described in the Experimental Procedures section of Example l- Each bar represents the mean +/- SD of triplicate determinations. Figure 5. Reactivity of anti-E-Selectin MAbε with Mutant Chimeras. E-Selectin-IgG chimeras with the mutations shown were tested for capture by each of fifteen anti-E-Selectin MAbs using the ELISA format described in the Experimental Procedures section of Example 1. Results shown represent the mean +/- S.D. of duplicate determinations and are expressed as percentage of control or wild type binding. Figure 6- Binding of Alanine Scan Mutants to Immobilized sLex Glycolip d. (A) E-Selectin-IgG chimeras in which the indicated residues were mutated to alanine were tested for binding to immobilized 2,3 sLex glycolipid by the ELISA procedure described in the Experimental Procedures section of Example 1. Results shown represent the mean +/- S.D. of triplicate determinations expressed as percentage of control or wild type binding. (B) E-Selectin-IgG mutant E8A (open squares) or wild type E-Selectin-IgG (closed squares) were tested at the indicated concentrations for binding to immobilized 2,3 sLex glycolipid by ELISA as above. Results shown repreεent the mean +/- S.D. of triplicate determinations. Figure 7. A Model of the Lectin Domain of E-Selectin- Shown is a ribbon model of the E-Selectin lectin domain derived from the published coordinates of the related type C lectin, the mannose binding protein [Weis et al.. Science 254. 1608-1615 (1991)] . Orientation A shows the amino acid residues whose mutation did not affect sLex or Mab binding (brown) , the residue at position 74 whose mutation did not affect sLex binding but did affect the binding of a number of non-blocking Mab (pink), the residues at positions 7,9 and 98 whose mutation abolished binding of blocking Mab (red) , the residues at positions 97,99 and 113 whose mutation abolished sLex binding (yellow) , and the residue at position 8 whose mutation enhanced the affinity of E-Selectin for sLex (green) . As noted hereinafter, mutation of residues 8 and 113 also affected the binding of some blocking Mabs. The single bound calcium is depicted as a green ball. Also shown in orientation A is the solution structure of sLex [Tyrrell et al., Proc. Natl. Acad- Sci. USA- 88, 10372-10376 (1991) ; Berg et al., J. Biol. Chem. 265. 14869-14872 (1991)] . The dark purple loop (residues S43-Y48) and the dark blue loop (residues Y94-D100) denote two loops near the carbohydrate binding site of E- Selectin that are not found in the mannose binding protein. Orientation B εhowε the "active site" of carbohydrate binding from a face-on view, with residues involved with carbohydrate recognition and/or blocking Mab binding colored as in orientation A. Figure 8. Binding of L-Selectin-IgG mutant K8A to immobilized sLex. L-Selectin-IgG mutant K8A (open squares) or wild type L-Selectin-IgG (closed squares) was tested at the indicated concentrations for binding to immobilized 2,3 sLex glycolipid by ELISA as above. Results shown represent the mean +/- S.D. of triplicate determinations. Figure 9. Structures of the εelectin (LEC-CAM) family memberε as determined by cDNA cloning. Illustrated are the εtructureε for L- Selectin, E-Selectin and P-Selectin. The lectin, epidermal growth facto (EGF) , and multiple εhort consensus repeats (SCRs) are shown wit hypothetical disulfide bond structures as first proposed for GMP-140 b Johnston et al . , Cell 56. 1033 (1989). An N-terminal sequence is als shown (subεequently cleaved in the mature protein) as well as hydrophobic transmembrane spanning anchor (TM) and cytoplasmic tail. Tw other forms of P-Selectin are also illustrated, one with a deleted ser- domain and another with a deleted membrane spanning anchor. Figure 10. Staining of HL-60 cells and neutrσphils by selectin-Ig chimeras. Selectin-IgG chimeras were tested by flow cytometry fo staining of either HL60 cells (A and C) or human neutrophils (B and D) aε described in Example 3. In (A) and (B) , solid lines represent P Selectin-IgG staining, doted lines represent P-Selectin-IgG staining i the presence of 10 mM EGTA, and dashed lines represent E-Selectin-Ig staining. Staining with no chimera (secondary antibody only) wa identical to E-Selectin-IgG staining for both cell types. (C) HL60 cell were treated as indicated in Example 3, and stained with P-Selectin-Ig as in (A) . Results are expressed as percentage of cells stainin positively (+/- SD of duplicates) based on staining with secondar antibody alone. (D) Human neutrophils before (solid bars) or after (ope bars) activation with PMA were εtained with the indicated reagents an evaluated by flow cytometry as deεcribed in Example 3. Reεults are show as the linear mean fluorescence. Figure 11. Binding of the PE-1 chimera to Immobilized sLe Glycolipids and Sulfatides. P-Selectin-IgG (open circleε) , E-Selectin IgG (open εquares) or PE-1 (closed circles) were tested at the indicate concentrations for binding to immobilized 2'3 sLex glycolipid (A), 2' sLex glycolipid (B) , or sulfatideε (C) by the ELISA procedure describe in Example 3. Results shown represent the mean +/- SD of triplicat determinations. Figure 12. Staining of HL60 cells with the PE-1 Chimera. The P Selectin-IgG, E-Selectin-IgG, and PE-1 chimeras were tested for bindin to HL60 cells by flow cytometry as in Figure 10. The percentage of cell staining positively (based on staining with the εecondary antibody alone) with each chimera iε indicated. Figure 13. Antibody Capture and 2'3 εLex binding of N82D E- Selectiή. N82D E-Selectin was generated and tested as described in Example 1. (a) Antibody capture; (B) binding to 2'3 sLex; (C) binding to 2'3 εLex in the preεence of added calcium- Figure 14- Reactivity of anti-P-Selectin mAb'ε with Mutant Chimeraε. P-Selectin-IgG chimeraε with the substitutions indicated were tested for capture by the mAb's AK-6 (solid bars), AC 1.2 (open bars), and CRC 81 (hatched bars) as in Table 1. Resultε εhown repreεent the mean +/- SD of duplicate determinations and are expressed as percentage of control P-Selectin-IgG binding. Figure 15. Binding of P-Selectin-IgG Mutantε to Immobilized Glycolipidε and Cells. P-Selectin-IgG chimeras with the substitutions indicated were tested for binding to immobilized 2'3 sLex glycolipid (A) , 2'6 sLex glycolipid (B) , or εulfatides (C) as in Figure 11, and for staining of HL60 cells (D) as in Figure 10. DETAILED DESCRIPTION OF THE INVENTION I. Definitions The term "selectin" " is used to refer to a cell adhesion molecule having, within its extracellular region, a lectin domain, an egf-like domain and complement binding-like domains and having the qualitative ability to mediate cell binding through interaction with a cell surface carbohydrate ligand, but nonetheless may have fewer or more domains if it still functions as a mediator of cell adhesion. The definition specifically includes "soluble" εelectin moleculeε lacking a functional transmembrane domain as well as amino acid sequence and glycosylation variantε and covalent modificationε of a native εelectin. The expression "native selectin" is used to define a native sequence selectin molecule of any human or non-human animal species including naturally occurring allelic variations that might occur from individual to individual, demonstrated by (an) amino acid difference(ε) in the overall sequence, without any limitation as to its mode of preparation- Thus, the native selectin may be obtained from any native source, may be produced synthetically or by recombinant DNA technology, or by suitable combination of such methods. The term specifically includes native mammalian, e.g. human and E-, L- and P-Selectins. The expression "corresponding selectin" as used throughout the specification and claimε referε to a εelectin molecule having an unaltered native selectin lectin domain, that may, however, have alterations in other parts of the moleculeε in agreement with the foregoing definition for "selectin". Thus, for example, for E-Selectin variants the corresponding εelectin has a native sequence (unaltered) E- Selectin lectin domain of any animal species but may otherwise differ from naturally occurring E-Selectins. In-the claims and throughout the specification certain alterations are defined with reference to amino acid residue numbers of the lectin domain of the corresponding native human selectin. The amino acid numbering starts at the first N-terminal amino acid of the lectin domain of a native human E-, L- or P-Selectin amino acid sequence. The amino acid sequence of the lectin domain of native human E-Selectin is shown in Figure 2(A) (amino acids 1-120). The lectin domain of native human L-Selectin compriseε amino acid residue numbers designated as residues 39-155 in Figure 1 of U.S. Patent No. 5,098,833 isεued 24 March 1992. Following the εequence numbering applied herein, theεe reεidues are referred to as amino acid residue numbers 1-117. The amino acid sequence of the lectin domain of P-Selectin is, for example, shown in Figure 2 of Lasky, L.A., J. Cell. Biochem. 45., 139-146 (1991) . The latter publication also shows the relative sequence homologies of the lectin domains.of the three εelectinε. The termε "amino acid" and "amino acids" refer to all naturally occurring L-α-amino acidε. This definition is meant to include norleucine, ornithine, and homocysteine. The amino acidε are identified by either the εingle-letter or three-letter designations: These amino acids may be clasεified according to the chemical compoεition and properties of their side chains. They are broadly classified into two groups, charged and uncharged. Each of these groups is divided into subgroups to clasεify the amino acids more accurately: I. Charged Amino Acids Acidic Residues: aspartic acid, glutamic acid Basic Residues: lysine, arginine, histidine II. Uncharged Amino Acids Hvdrophilic Residues: serine, threonine, asparagine, glutamine Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine Non-polar Residues: cysteine, methionine, proline Aromatic Residues: phenylalanine, tyrosine, tryptophan The terms "amino acid alteration" and "alteration" refer to amino acid substitutions, deletions or insertions or any combinations thereof in a εelectin amino acid εequence. In the selectin variants of the preεent invention εuch alteration iε at a εite or εiteε of a selectin lectin domain amino acid sequence- Substitutional variants herein are those that have at least one amino acid residue in a native εelectin lectin domain sequence removed and a different amino acid inserted in its place at the same position. The εubstitutionε may be εingle, where only one amino acid in the molecule haε been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. Inse tional variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native selectin lectin domain sequence- Immediately adjacent to an amino acid means connected to either the or-carboxy or α-amiπo functional group of the amino acid. The insertion may be one or more amino acids. Ordinarily, the insertion will consist of one or two conservative amino acids. Amino acids similar in charge and/or structure to the amino acids adjacent to the site of insertion are defined as conservative. Alternatively, this invention includes insertion of an amino acid with a charge and/or structure that iε εubstantially different from the amino acids adjacent to the site of insertion. Deletional variants are those with one or more amino acidε in the native εelectin lectin domain amino acid εequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the lectin domain amino acid sequence. The designation of the substitution variants herein coπsiεts of a letter followed by a number followed by a latter. The firεt (leftmost) letter designates the amino acid in the native (unaltered) selectin lectin domain. The number refers to the amino acid position where the amino acid substitution is being made, and the second (righthand) letter deεignateε the amino acid that is used to replace the native amino acid. The designation for an insertion variant consists of the letter "i" followed, by a number designating the position of the residue in a native selectin amino acid sequence before which the insertion starts, followed by one or more capital letters indicating, inclusively, the insertion to be made. The designation for a deletion variant consistε of the letter "d" followed by the number of the εtart poεition of the deletion to the number of the end position of the deletion, with the positions being based on the amino acid sequence of the native sequence, unaltered lectin domain of the corresponding (E, L or P) selectin- As mentioned before, the numbering starts at the N-terminal amino acid sequence of the selectin lectin domain (which is the N-terminal end of the mature native selectin molecule) . Multiple alterations are separated by a comma (,) in the notation for ease of reading them. The termε "nucleic acid molecule encoding", "DNA sequence encoding", and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The -order of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain. The DNA εequence thuε codes for the amino acid sequence. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to a DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding εequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptorε or linkerε are used in accord with conventional practice. The terms "replicable expresεion vector" and "expreεεion vector" refer to a piece of DNA, uεually double-stranded, which may have inserted into it a piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA may be generated. In addition, the vector contains the necesεary elementε that permit tranεlating the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized. In the context of the present invention the expressions "cell", "cell line", and "cell culture" are used interchangeably, and all εuch designations include progeny. It is also understood that all progeny ma not be precisely identical in DNA content, due to deliberate o inadvertent mutations. Mutant progeny that have the same function o biological property as screened for in the originally transformed cel are included. "Transformation" means introducing DNA into an organism so that th DNA is replicable, either as an extrachromosomal element or b chromosomal integration. "Transfection" referε to the taking up of an expression vector by a host cell whether or not any coding εequenceε are in fact expreεεed. The terms "transformed host cell ,r and "transformed" refer to the introduction of DNA into a cell. The cell is termed a "host cell", and it may be a prokaryotic or a eukaryotic cell- Typical prokaryotic host cells include various strains of E.. coli- Typical eukaryotic host cells are mammalian, such as Chinese hamster ovary cells or human embryonic kidney 293 cells. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DNA sequence may be from the εame εpecies as the host cell or a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign and some homologous DNA. "Oligonucleotides" are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods [such as phosphotriester, phosphite, or phosphoramidite chemiεtry, using solid phase techniques such as those described in EP 266,032, published 4 May 1988, or via deoxynucleσside H-phosphanate intermediates as described by Froehler et al. , Nucl. Acids Res. 14, 5399 (1986)] - They are then purified on polyacrylamide gels. The technique of "polymerase chain reaction" or "PCR", aε uεed herein, generally referε to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Patent No. 4,683,195, issued 28 July 1987 and in Current Protocols in Molecular Biology. Ausubel et al. eds., Greene Publishing Associateε and Wiley-Interscience 1991, Volume 2, Chapter 15. The term "monoclonal antibody" as used herein re ers to an antibody obtained from a population of subεtantiallyhomogeneouε antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be preεent in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. The monoclonal antibodies include hybrid and recombinant antibodies produced by splicing a variable (including h pervariable) domain of an anti-εelectin ligand antibody with a constant domain (e.g. "humanized" antibodies) , only one of which is directed against a selectin, or a light chain with a heavy chain, or a chain from one εpecieε with a chain from another εpecies, or fuεions with heterologous proteins, regardless of species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g.. Fab, F(ab'),, and Fv) . Cabilly, et al., U.S. Pat. No- 4,816,567; Mage --. Lamoyi, in Monoclonal Antibody Production Techniques and Applications, pp.79-97 (Marcel Dekker, Inc., New York, 1987). Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from such a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The expression "blocking monoclonal antibody" is used to refer to monoclonal antibodies capable of inhibiting the binding of a selectin variant of the present invention to a native ligand of the corresponding (E, L or P) selectin in a standard binding aεεay such as the asεay deεcribed in Example 1. The term "immunoglobulin" generally refers to polypeptides comprising a light or heavy chain usually both disulfide bonded in the native "Y" configuration, although other linkage between them, including tetramers or aggregates thereof, is within the εcope hereof. Immunoglobulins (Ig) and certain variants thereof are known and many have been prepared in recombinant cell culture. For example, see U.S. Patent 4,745,055; EP 256,654; Faulkner et al., Nature 291:286 (1982) ; EP 120,694; EP 125,023; Morrison, J. Immun. 121:793 (1979); Kόhler et al., Proc. Nat'l. Acad. Sci. USA 22:2197 (1980); Raεo et al., Cancer Reε. 11:2073 (1981); Morrison et al . , Ann. Rev. Immunol. 2_:239 (1984); Morriεon, Science 229:1202 (1985); Morrison et al., Proc. Na '1. Acad. Sci. USA £1:6851 (1984); EP 255,694; EP 266,663; and WO 88/03559. Reasεorted immunoglobulin chainε also are known. See for example U.S. patent 4,444,878; WO 88/03565; and EP 68,763 and references cited therein. L-Selectin-immunoglobulin chimeras are, for example, disclosed in WO 91/08298 published 13 June 1991. The production and characterization of E-Selectin-immunoglobulin chimeras has been reported by Foxall et al . , J. Cell. Biol. 1992, in press. P-Selectin- immunoglobulin chimeras have been constructed in an analogous manner. The immunoglobulin moiety in the chimera of the present invention may be obtained from IgG,, IgG 2 , lgG 3/ or IgG 4 subtypes, IgA, IgE, IgD or IgM, but preferably IgG, or IgG 3 . II. Construction of the Selectin Variants and their Derivatives Site Directed Mutagenesis Preparation of selectin variants in accordance herewith is preferably achieved by site-specific mutagenesis of DNA that encodes an earlier prepared variant or a nonvariant version of the protein. Site- εpecific mutageneεis allows the production of selectin variantε through the uεe of specific oligσnucleotide sequenceε that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides to provide a primer εequence of εufficient size and sequence complexity to form a stable duplex on both sides of the junction being traversed. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the εequence being altered. In general, the technique of site-εpecific mutageneεis is well known in the art as exemplified by publications such as Adelman et al. , DNA, 2 : 183 (1983) . As- will be appreciated, the site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutageneεis include vectorε such aε the M13 phage, for example, as disclosed by Messing et al. , Third Cleveland Symposium on Macromolecules and Recombinant DNA. Editor A. Walton, Elsevier, Amsterdam (1981) . These phage are readily commercially available and their use is generally well known to those skilled in the art. Alternatively, plasmid vectors that contain a single-stranded phage origin of replication (Veira et al-, Meth. Enzvmol.. 153: 3 (1987) ) may be employed to obtain single-stranded DNA. The specific mutagenesis method followed in making the E-Selectin variants of Example 1 was deεcribed by Kunkel et al., Methodε in Enzvmol. 154367-382 (1987) . In general, site-directed mutagenesis may, for example, be performed by first obtaining a single- tranded vector that includes within its sequence a DNA sequence that encodes the relevant εelectin. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al. , Proc. Natl. Acad. Sci- (USA) , 2ϋ-: 5765 (1978) . This primer is then annealed with the single-stranded selectin sequence-containing vector, and subjected to DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the syntheεiε of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second εtrand bearε the desired mutation. This heteroduplex vector is then used to transform appropriate cells such as JM101 cells and clones are selected, via hybridization to a radioactive probe consisting of the 32 P-labeled mutageneεiε primer, that include recombinant vectorε bearing the mutated sequence arrangement. After such a clone is selected, the mutated selectin region may be removed and placed in an appropriate vector for selectin production, generally an expression vector of the type that typically is employed for transformation of an appropriate eukaryotic host. In the context of the present invention, Chinese hamster ovary (CHO) cells or 293 (human kidney cells described by Graham et al. r J. Gen. Virol.. 36: 59 (1977)) are preferred for the preparation of long-term stable selectin producers. However, the invention iε not limited to CHO production, as it is known that numerous other cell types are suitably employed, particularly where one desires only transient production of the enzyme for test purposes. For example, described below is a transient syεte employing 293 cells that provides a convenient system for production of selectin variants for analytical purposes- Another method for making mutations in the DNA εequence encoding a selectin involves cleaving the DNA at the appropriate position by digeεtion with restriction enzymes, recovering the properly cleaved DNA, synthesizing an oligonucleotide encoding the desired amino acid and flanking regions such as polylinkers with blunt ends (or, instead of using polylinkers, digesting the synthetic oligonucleotide with the restriction enzymes also used to cleave the selectin-encoding DNA, thereby creating coheεive termini) , and ligating the synthetic DNA into the remainder of the selectin-encoding structural gene. PCR Mutagenesis PCR mutageneεis is also suitable for making the selectin amino acid variants of the present invention. While the following diεcussion referε to DNA, it is understood that the technique also find application with RNA. The PCR technique generally refers to the following procedure. When small amounts of template DNA are used as starting material in PCR, pri erε that differ εlightly in εequence from the correεpondin region in a template DNA can be used to generate relatively larg quantities of a specific DNA fragment that differs from the templat sequence only at the positionε where the primerε differ from th template. For introduction of a mutation into a plasmid DNA, one of th primers is designed to overlap the position of the mutation and t contain the mutation; the sequence of the other primer muεt be identica to a stretch of sequence of the opposite strand of the plasmid, but thi sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 20 nucleotides from that of the first, such that in the end the entir amplified region of DNA bounded by the primerε can be easily εequenced PCR amplification using a primer pair like the one just described result in a population of DNA fragmentε that differ at the poεition of th mutation specified by the primer, and possibly at other positions, a template copying is somewhat error-prone. If the ratio of template to product material is extremely low, th vast majority of product DNA fragmentε incorporate the deεire mutation(ε) . Thiε product material iε used to replace the correspondin region in the plasmid that served as PCR template using εtandard DN technology. Mutationε at separate positions can be introduce simultaneously by either using a mutant εecond primer or performing εecond PCR with different mutant primerε and ligating the two reεultin PCR fragmentε simultaneously to the vector fragment in a three (or more) part ligation. Host Cell Cultures and Vectors Although expression on Chinese hamster ovary (CHO) cellε and in th human embryonic kidney cell line 293 [Urlaub and Chasin, Proc. Natl Acad. Sci. USA 22 » 4216 (1980); Graham et al., . Gen. Virol.. 36, 5 (1977) ] are ultimately preferred for the production of the selecti variants herein, the vectors and methods disclosed herein are suitabl for use in host cells over a wide range of eukaryotic organisms. In general, of course, prokaryotes are preferred for the initia cloning of DNA sequences and constructing the vectors useful in th invention. For example, E ^ . coli K12 strain 294 (ATCC No. 31,446) and E. coli strain W3110 (ATCC No- 27,325) are particularly uεeful. Other εuitable microbial strains include E_-. coli strainε εuch as J - coli B, and E- coli X1776 (ATCC No. 31,537) . These examples are, of course, intended to be illustrative rather than limiting. Prokaryotes also are useful for expression- The aforementioned strains, as well as bacilli such aε Bacillus subtiliε. and other enterobacteriaceae εuch as, e.g., Salmonella tvphimurium or Serratia marcesanε. and various Pseudomonas εpecieε are exampleε of uεeful hosts for expression. In ' general, plasmid vectors containing replicon and control sequenceε that are derived from εpecies compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences that are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed uεing pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al.. Gene. 2.: 95 (1977))- pBR322 contains genes for ampicillin and tetracycline resiεtance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage, must also contain, or be modified to contain, promoters that can be used by the microbial organism for expression of its own proteins- Thoεe promoters most commonly used in recombinant DNA construction include β-lactamase (penicillinase) and lactose promoter systems (Chang et al.. ' Nature. 375: 615 (1978); Itakura et al., Science, 198: 1056 (1977); Goeddel et al., Nature. 281: 544 (1979)) and a tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res.. £: 4057 (1980); EPO Appl. Publ. No. 36,776), and the alkaline phosphatase syεtemε. While theεe are the moεt commonly uεed, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (see, e.g., Siebenlist et al., Cell. 20.: 269 (1980)) . In addition to prokaryotes, eukaryotic microbes, such as yeasts, also are suitably used herein. Saccharomvces cerevisiae. or common baker's yeast, is the moεt commonly uεed among eukaryotic microorganiεmε, although a number of other strains are commonly available- For example, for expression in Saccharomvces. the plasmid YRp7 (Stinchcomb et al-, Nature. " 282: 39 (1979); Kingsman et al., Gene. 2= 141 (1979); Tschemper et al.. Gene- 10: 157 (1980)) is commonly used. Thiε plaεmid already containε the trpl gene that provideε a εelection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44,076 or PEP4-1 (Jones, Genetics. 85: 12 (1977)) . The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al. , J. Biol . Chem. , 255 : 2073 (1980)) or other glycolytic enzymes (Hess et al. , J. Adv. Enzyme Reg., : 149 (1968); Holland et al. , Biochemistry. 17: 4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinaεe, pyruvate decarboxylaεe, phoεphofructokinaεe, glucoεe-6-phoεphate iεomeraεe, 3- phoεphoglycerate mutaεe, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinaεe. In the. conεtruction of suitable expression plasmids, the termination sequences asεociated with theεe geneε are alεo ligated into the expreεsion vector 3' of the εequence deεired to be expreεεed to provide polyadenylation of the mRNA and termination. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes' associated with nitrogen metabolism, and the aforementione glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible fo maltose and galactose utilization. Any plaεmid vector containing yeast- compatible promoter, origin of replication and termination εequences i εuitable. In addition to microorganiεmε, cultures of cells derived fro multicellular organisms may also be used aε hosts. In principle, an such cell culture is workable, whether from vertebrate or invertebrat culture. However, interest has been greatest in vertebrate cells, an propagation of vertebrate cellε in culture (tissue culture) has becom a routine procedure in recent years fTisεue Culture. Academic Preεs, Kruse and Patterson, editors (1973)] . Examples of such useful host cel lines are VERO and HeLa cellε, CHO cell lineε, and W138, BHK, COS-7, (ATCC CRL 1651), 293, and MDCK (ATCC CCL 34) cell lineε. Expreεsio vectors • for such cells ordinarily include (if necessary) an origin o replication, a promoter located in front of the gene to be expresεed along with any necessary ribosome binding siteε, RNA εplice sites polyadenylation sites, and transcriptional terminator εequences. For use in mammalian cells, the control functions on the expressio vectors are often provided by viral material. For example, commonly use promoters are derived from polyoma, Adenovirus2, and moεt frequentl Simian Viruε 40 (SV40) . The early and late promoters of SV40 virus ar particularly useful because both are obtained easily from the virus a a fragment that also contains the SV40 viral origin of replication (Fier et al., Nature, 273: 113 (1978)) . Smaller or larger SV40 fragments ar also suitably used, provided there is included the approximately 250-b sequence extending from the HindiII εite toward the Bgll site located i the viral origin of replication. Further, it is also posεible, and ofte deεirable, to utilize promoter or control sequences normally associate with the desired gene sequence, provided such control sequences ar compatible with the host cell syεtems- An origin of replication typically is provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or by the host cell chromoεomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient. Satisf ctory amounts of human εelectin variants are produced by cell cultures; however, refinements, uεing a εecondary coding εequence, serve to enhance production levels even further- The secondary coding sequence comprises dihydrofolate reductase (DHFR) that is affected by an externally controlled parameter, such as methotrexate (MIX) , thus permitting control of expression by control of the MTX concentration. In the selection of a preferred host cell for transfection by the vectors of the invention that comprise DNA sequences encoding both variant selectin and DHFR protein, it is appropriate to consider the type of DHFR protein employed. If wild-type DHFR protein is employed, it is preferable to select a host cell that is deficient in DHFR, thus permitting the use of the DHFR coding sequence as a marker for succeεsful transfection in selective medium that lacks hypoxanthine, glycine, and thymidine. An appropriate host cell in this case is the CHO cell line deficient in DHFR activity, prepared and propagated, as described by Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 22: 4216 (1980) . On the other hand, if DHFR protein with low binding affinity for MTX is used as the controlling sequence, it is not necessary to use DHFR- deficient cellε. Becauεe the mutant DHFR is resistant to MTX, MTX- containiπg media can be used as a means of selection, provided that the host cells are themselves MTX sensitive. Most eukaryotic cells that are capable of absorbing MTX appear to be sensitive to MTX. One εuch uεeful cell line is a CHO line, CHO-K1 (ATCC No. CCL 61) . Typical Cloning and Expresεion Methodoloσieε Available If mammalian cellε are used as host cellε, transfection generally is carried out by the calcium phosphate precipitation method as described by Graham and Van der Eb, Virology. 52: 546 (1978) . However, other methods- for introducing DNA into cellε such aε nuclear injection, electropora ion, or protoplast fusion are also suitably used. If yeast are used aε the host, transfection iε generally accompliεhed uεing polyethylene glycol, as taught by Hinnen, Proc- Natl. Acad- Sci. U.S.A., 25: 1929-1933 (1978). If prokaryotic cells or cells that contain substantial cell wall constructions are used, the preferred method of transfection is calcium treatment using calcium as described by Cohen et al. , Proc. Natl- Acad. Sci. (USA) 69.: 2110 (1972) , or more recently electroporation. Construction of suitable vectors containing the desired coding and control sequences employs standard ligation techniques. Isolated plasmidε or DNA fragmentε are cleaved, tailored, and religated in the form desired to form the plasmids required. Cleavage is performed by treating with restriction enzyme (or enzymes) in suitable buffer. In general, about 1 μg plasmid or DNA fragments is used with about 1 unit of enzyme in about 20 μl of buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer.) Incubation times of about one hour at 37°C are workable. After incubation, protein is removed by extraction with phenol and chloroform, and the nucleic acid is recovered from the aqueous fraction by precipitation with ethanol. If blunt ends are required, the preparation may be treated for 15 minutes at 15°C with 10 units of the Klenow fragment of DNA Polymeraεe I (Klenow) , phenol-chloroform extracted, and ethanol precipitated. Size εeparation of the cleaved fragments is performed using 6 percent polyacrylamide gel described by Goeddel et al. , Nucleic Acids Res.. : 4057 (1980) . For ligation, approximately equimolar amounts of the deεire components, suitably end tailored to provide correct matching, are treated with about 10 units T4 DNA ligaεe per 0.5 μg DNA. (When cleave vectors are used as components, it may be useful to prevent religatio of the cleaved vector by pretreatment with bacterial alkalin phosphatase.) As discusεed above, selectin variants are preferably produced b means of specific mutation. Variants useful in the practice of th present invention are formed most readily through the use of specifi oligonucleotide sequences that encode the DNA sequence of the desire mutation, as well as a sufficient number of adjacent nucleotides, t provide a sequence of sufficient size and sequence complexity to form stable duplex on both sides of the mutation being traversed. For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are typically used to transform E^. coli K12 (ATC 31,446) or other suitable E ^ . coli strains, and successful tranεformant selected by ampicillin or tetracycline resistance where appropriate Plasmids from the transformants are prepared and analyzed by reεtrictio mapping and/or DNA sequencing by the method of Messing et al. , Nuclei Acids Res.. 9.: 309 (1981) or by the method of Maxam et al., Methods o Enzvmology, 65: 499 (1980) . After introduction of the DNA into the mammalian cell host an selection in medium for stable transformantε, amplification of DHFR protein-coding sequences is effected by growing host cell cultures in th presence of approximately 20,000-500,000 nM concentrations of MTX, competitive inhibitor of DHFR activity. The effective range o concentration is highly dependent, of courεe, upon the nature of the DHF gene and protein and the characteristics of the host. Clearly, generall defined upper and lower limits cannot be ascertained. Suitabl concentrations of other folic acid analogs or other compounds that inhibit DHFR could also be used. MTX itself is, however, convenient, readily available, and effective- Glycosylation Variants Glycosylation of polypeptides is typically either N-linked or 0- linked. N-linked refers to the attachment of the carbohydrate moiety to the side-chain of an asparagine residue- The tripeptide εequences, asparagine-X-εerine and asparagine-X-threonine, wherein X is any amino acid except proline, are recognition εequenceε for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. O-linked glycosylation referε to the attachment of one of the εugarε N- acetylgalactosamine, galactoεe, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5- hydroxylysine may alεo be involved in O-linked glycosylation- O-linked glycoslation sites may, for example, be modified by the addition of, or substitution by, one or more serine or threonine residue to the amino acid sequence of the ligand. For ease, changes are usually made at the DNA level, essentially using the techniques discussed hereinabove with respect to the amino acid sequence variants. Chemical or enzymatic coupling of glycosydes to the selectin variants of the present invention may also be used to modify or increase the number or profile of carbohydrate substituents. These procedures are advantageous in that they do not require production of the polypeptide that is capable of O-linked (or N-linked) glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free hydroxyl groups such as those of cysteine, (d) free εulfhydryl groups εuch aε those of serine, threonine, or hydroxyproline, (e) aromatic residueε εuch aε those of phenylalaniπe, tyrosine, or tryptophan or (f) the amide group of glutamine. These methods are described in WO 87/05330 (published 11 September 1987) , and in Aplin and Wriεton, CRC Crit. Rev. Biochem.. pp. 259-306 (1981) . Carbohydrate moietieε present on a εelectin variant may also be removed chemically or enzymatically. Chemical deglycosylation requires exposure to trifluoromethanesulfonic acid or an equivalent compound. This treatment reεultε in the cleavage of moεt or all sugars, except the linking sugar, while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al. , Arch- Biochem. Biophvs. 259. 52 (1987) and by Edge et al., Anal. Biochem. 118. 131 (1981) . Carbohydrate moieties can be removed by a variety of eπdo- and exoglycosidases aε described by Thotakura et al. , Meth. Enzvmol. 138. 350 (1987) . Glycosylation is suppreεεed by tunicamycin aε described by Duskin et al., J. Biol. Chem. 257- 3105 (1982) . Tunicamycin blocks the formation of protein-N-glycosydaεe linkages- Glycosylation variants of the variants herein can alεo be produced by selecting appropriate host cells. Yeast, for example, introduce glycosylation which varies significantly from that of mammalian systems. Similarly, mammalian cells having a different species (e.g. hamster, murine, insect, porcine, bovine or ovine) or tissue (e.g. lung, liver, lymphoid, mesenchymal or epidermal) origin than the source of the selectin variant, are routinely screened for the ability to introduce variant glycosylation. Covalent Modifications Covalent modifications of a selectin variant molecule are included within the scope herein. Such modifications are traditionally introduced by reacting targeted amino acid residueε of the εelectin variant with an organic derivatizing agent that iε capable of reacting with εelected side-chains or terminal residues, or by harnesεing mechaniεmε of poεt- translational modifications that function in selected recombinant host cells. ' The resultant covalent derivativeε are useful in programs directed at identifying residues important for biological activity, for immunoassays of the εelectin ligandε, or for the preparation of anti- εelectin ligand antibodieε for immunoaffinity purification of the recombinant glycoprotein. For example, complete inactivation of the biological activity of the protein after reaction with ninhydrin would εuggest that at least one arginyl or lysyl residue is critical for its activity, whereafter the individual residues which were modified under the conditions εelected are identified by isolation of a peptide fragment containing the modified amino acid residue. Such modifications are within the ordinary skill in the art and are performed without undue experimentation. Derivatization with bifunctional agents is useful for preparing intramolecular aggregates of the selectin variants as well as for cross- linking' the εelectin variantε to a water insoluble support matrix or surface for use in aεεayε or affinity purification. In addition, a study of interchain crosε-links will provide direct information on conformational structure. Commonly used cross-linking agents include 1,1-bis(diazoacetyl) -2-phenylethane,glutaraldehyde,N-hydroxyεuccinimide esters, homobifunctional imidoesterε, and bifunctional maleimides. Derivatizingagentε suchas methyl-3- [(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates which are capable of forming cross¬ links in the presence of light. Alternatively, reactive water insoluble matrices such as cyanogen bromide activated carbohydrates and the systems reactive substrates described in U.S. patent Nos. 3,959,642; 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employed for protein immobilization and cross-linking. Certain post-translational modifications are the result of th action 'of recombinant host cells on the expressed polypeptide. Glutaminyl and aspariginyl residues are frequently post-translationall deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions- Either form of these residues falls within the scope of this invention. Other post-tranεlational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the αr-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties. W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)] . Other derivatives comprise the novel εelectin variants of this invention covalently bonded to a nonproteinaceous polymer. The nonproteinaceous polymer ordinarily iε a hydrophilic εynthetic polymer, i.e. a polymer not otherwiεe found in nature. However, polymerε which exiεt in nature and are produced by recombinant or in vitro methodε are uεeful, aε are polymerε which are isolated from nature- Hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularly useful are polyvinylalkylene ethers εuch a polyethylene glycol, polypropylene glycol. The selectin variants may be linked to various nonproteinaceous polymerε, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The selectin variants may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery syεtemε (e.g. liposomeε, albuminmicrosphereε, microemulsions, nano-particles and nanocapsules) , or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences. 16th Edition, Osol, A-, Ed. (1980)- Construction of Selectin Variant-Immunoglobulin Chimeras A selectin variant sequence can be linked to a immunoglobulin constant domain sequence aε hereinbefore defined. The resultant moleculeε are commonly referred to aε εelectin immunoglobulin chimeras. Such chimeras can be constructed essentially as described in WO 91/08298 (published 13 June 1991) . Ordinarily, the εelectin variant is fused C-terminally to the N- terminus of the constant region of an immunoglobulin in place of the variable region(ε) , however N-terminal fuεionε of the εelectin variantε are also desirable. The transmembrane regions of the selectin variants are preferably inactivated or deleted prior to fusion. Typically, such fusions retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobuli.n heavy chain- Fuεionε are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CHI of the heavy chain or the corresponding region of the light chain. This ordinarily is accomplished by constructing the appropriate DNA sequence and expreεεing it in recombinant cell culture. Alternatively, however, the εelectin variant-immunoglobulin chimeraε of this invention may be synthesized according to known methods. The precise εite at which the fusion iε made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion or binding characteristics of the selectin variant. In some embodiments, the hybrid immunoglobulins are assembled as monomers, or hetero- or homo-multimers, and particularly as dimers of tetramers, essentially aε illuεtrated in WO 91/08298, Supra. In a preferred embodiment, the C-terminuε of a sequence which containε the binding εite(ε) for a selectin ligand, is fused to the N- terminus of the C-terminal portion of an antibody (in particular the Fc domain) , containing the effector functions of an immunoglobulin, e.g. immunoglobulin G, . It is possible to fuse the entire heavy chain constant region to the sequence containing the ligand binding site(ε) . However, more preferably, a εequence beginning in the hinge region just upstream of the papain cleavage site (which defineε IgG Fc chemically; residue 216, taking the first residue of heavy chain constant region to be 114 [Kobet et al., Supra] . or analogous εiteε of other immunoglobulins) is used in the fusion. In a particularly preferred embodiment, the amino acid sequence containing the ligand binding εite(s) (preferably retainin the egf like and complement binding domainε) is fused to the hinge regio and C H 2 and C H 3 or C H 1, hinge, C H 2 and C H 3 domains of an IgG,, IgG- or IgG heavy chain. The precise site at which the fusion is made is no critical, and the optimal site can be determined by routin experimentation. III. Preferred Selectin Variants Ordinarily, the selectin variants of the present invention will hav a lectin domain the amino acid sequence of which is substantiall homologous (ordinarily more than about 80% based on complete amino aci identity, ignoring insertionε or deletionε) to that of a native εelecti molecule, but containing one or more amino acid residue substitutions, deletions or insertions at one or more of certain specified sites withi this domain. The variant selectin molecules of the present invention ma exhibit, altered (preferably enhanced) affinity for binding to corresponding native selectin ligand as a result of the alteration(s) i their lectin domains. The inventive amino acid sequence variants of th present invention are one or combination of substitutional, insertional or deletional variants at certain sites which have been identified by th inventors to be important or influential in modulating the ligand bindin properties of εelectinε. The amino acid reεidues identified aε primarily involved with recognition of the carbohydrate ligands of selectinε are a relatively small patch on the surface of the selectin lectin domain near the antiparallel beta sheet formed by the disulfide-linked N- and C-termini ' and the adjacent diεulfide linked loop formed by the two internal cyεteineε- Based upon experimental evidence disclosed in the Examples, it is believed especially that positively charged amino acid residues in this region are critical for carbohydrate recognition. Accordingly, alterations in this region, and especially at positively charged amino acids are expected to have the greatest influence on the ligand binding properties of the selectin molecule. In general, substantial changes in the ligand binding properties of the εelectin molecule may be obtained by substituting amino acids within the above-identified region which have significantly different side chains than the native residue at that site. Such substitutions would be expected to affect (a) the structure of the polypeptide backbone in the area of substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which would be expected to cause the greatest changes in the chemical and/or phyεical propertieε of the selectin molecule are those in which (a) a basic (positively charged) residue is substituted with an aliphatic or aromatic residue, (b) an acidic reεidue is substituted with an aliphatic or aromatic residue, (c) a hydrophilic residue is substituted with an aromatic or aliphatic residue, (d) an aromatic reεidue iε εubεtituted with an acidic or basic residue, (e) an aliphatic residue is εubεtituted with an acidic or basic residue, or (f) a non-polar reεidue is substituted with an acidic or basic resdue. In particular, three positively charged amino acid residues that proved to be critical for sLex recognition by E-Selectin, arginine (R) at residue number 97, lysine (K) at reεidue number 111, and lysine (K) at reεidue number 113, are situated very close to each other on the E- Selectin model shown in Figure 7. Lysine at amino acid positions 111 and 113 is conserved in all three selectins from a number of different species. Accordingly, the presence of a positively charged residue, e.g., lysine, at thiε position is believed to be essential for ligand binding. The replacement of thiε amino acid residue with an uncharged amino acid (alanine) completely abolished sLex recognition. The replacement of arginine at poεition 97 of the selectin lectin domain is conserved in E- and L-Selectin only, whereas P-Selectin has serine (a hydrophilic uncharged amino acid) at this site. In E-Selectin, the substitution of alanine for this residue abolished sLex binding, hence, this residue is alεo critical for carbohydrate recognition. An entirely unexpected finding waε the increaεe in ligand binding affinity observed as a result of the substitution of alanine for glutamic acid (a charged residue) at position 8 of the E-Selectin amino acid sequence. L- and P-Selectin contain a positive charge (K) at this site, the mutation of which to alanine in L-Selectin was found to significantly enhance sLex recognition (Example 2) . Accordingly, substantial changes in the ligand binding properties of a selectin molecule can be expected from alterations within the above- identified patch in the selectin lectin domain, and in particular, from replacing charged amino acid residues by uncharged residueε at the above- described positionε. In addition to the charge, the bulk of the amino acid iε an important consideration. Exemplary variant selectin molecules constructed as single-site substitutional variants of selectinε are liεted in Table 1 below. Modulating Site E8 (K8*) R97** Kill K113 * for L- and P-Selectin ** for E- and L-Selectin In addition to the single-site substi u ional variant selecti molecules listed in Table 1, the preferred multiple-site subεtitutiona variant moleculeε include thoεe having any of the foregoing amino acid substituted at position 8 of the selectin lectin domain, combined wit one or more of the foregoing alterations at positionε 97, 111 and 113. Similar subεtitutionε are poεεible in the amino acid regionε 7-9, 43-48, 82-86, and 90-109, and specifically at poεitionε 7, 8, 9, 47, 48, 82, 84, 86, 94, 96, 98 and 100, alone or in any combination. The variants herein preferably retain the egf-like domains and th complement binding domains of native selectinε, but may compris additional alterations (e.g., conservative amino acid εubstitutions) a other parts of the molecule without significantly affecting the ligan binding properties of the resultant variants. Additional alterations ar alεo poεεible at regionε of the lectin domain identified as not bein involved in ligand binding. The information disclosed herein about the ligand binding sites o selectins can be further refined by further mutagenesis studieε, such a by homolog-scanning and by high resolution alanine-scanning mutagenesi [Cunningham, B. C. et al. , Science 243 : 1330-1336 (1989); Cunningham B. C. and Wells, J.A., Science 24 : 1081-1085 (1989); for review se Wells, J.A., Methods Enzvmol. 202: 390-411 (1991)], and ultimately b determining the crystal structures of native selectins. IV. Therapeutic Compositions The selectin variants with enhanced ligand binding affinity can be used to block the binding of a corresponding selectin receptor to its native ligand. For example, an L-Selectin variant with enhanced ligand binding propertieε effectively blockε the binding of an L-Selectin receptor on a circulating leukocyte to itε native ligand on an endothelial cell. Thiε property is useful for treating a symptom or condition associated with excesεive binding of circulating leukocytes to endothelial cells, such aε inflammation aεsociated with rheumatoid arthritis, psoriasis, multiple sclerosis, etc. The selectin variants of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the ligand iε combined in admixture with a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. These compositionε will typically contain an effective amount of the selectin variant, for example, from on the order of about 0.5 to about 10 mg/ml, together with a suitable amount of carrier to prepare pharmaceutically acceptable compositions suitable for effective administration to the patient. The variants may be administered parenterally or by other methods that ensure its delivery to the bloodstream in an effective form. Compositions particularly well suited for the clinical administration of the selectin variants uεed to practice thiε invention include sterile aqueous solutions or εterile hydratable powders εuch aε lyophilized protein. Typically, an appropriate amount of a pharmaceutically acceptable salt is also used in the formulation to render the formulation isotonic. Dosages and desired drug concentrations of pharmaceutical compositions of this invention may vary depending on the particular use envisioned. Further details of the invention are illustrated in the following non-limiting Examples. V. Examples Example 1 A. Experimental Procedures Production and characterization of anti-E Selectin monoclonal antibodies Monoclonal antibodies to both human and rabbit E-Selectin were generated by immunization of mice with COS cells transiently expresεing E-Selectin. COS cells (5 x 10 7 /0.8 ml in Dulbecco's phosphate buffered saline (DPBS) were tranεfected by electroporation (350 V, 250 μF, Bio-Rad Gene Pulser) with 20 μg human or rabbit E-Selectin cDNA, incubated on ice for 10 min., resuspended in Dulbecco's modified Eagle's medium (DMEM) /10% fetal bovine serum (FBS) and plated at 10 7 cells/225 cm : tisεue culture flaεk. Transfected cells were harvested non-enzymatically at 48-72 hrs. , washed twice and resuεpended in DPBS. Mice were routinely immunized i.p. with 1 x 10 7 cells and boosted every 2-3 weeks. Hybridomas were produced by fuεion of immunized mouεe εplenocyteε with SP2/0 cellε using standard techniques [Galfre et al. Nature 266. 550-552 (1977)] . Hybridoma supernatantε were εcreened by a differential binding enzyme- linked immunoassay (ELISA) to Immulon 2 microtitre plates (Dynatech Laboratories, Inc., Chantilly, VA.) coated with detergent extracts of membranes from E-Selectin transfected and control/non-transfected COS cellε. Crude membrane fractionε were extracted in 2% Triton X-100, 150 mM NaCl, 50 mM Triε pH 7.5 (2.5 x 10 s cell equivalents/ml) . Extracts were diluted in 50 mM Na 2 C0 3 pH 9.6 and directly coated onto assay plates. Anti-human antibodies were also screened by ELISA assay for selective binding to human umbilical vein endothelial cellε (HUVECε) treated for 4 hrs with.rhlL-la (550 pg/ml) and rhTNF (400 U/ml) compared to untreated HUVECs. The εpecificity of theεe mAb's were confirmed with cytokine treated HUVECs, and transiently transfected COS cells by immunoblotting, immunoprecipitation, and indirect immunofluorescence (Wolitzky et al in preparation) . Mab 7H5 (IgG3), 8E4 (IgG2A) , 3B7 (IgGl) , 1D6 (IgGl) , 4D9 (IgG3) , 1E5 (IgGl) , 9A1 (IgGl) , 7E10 (IgGl) , and 1B3 (IgM) were generated to human E-Selectin, while MAb's 14G2 (IgGl), 11G5 (IgM), and 9H9 (IgM) were produced to rabbit-E-Selectin. Asciteε waε produced by εtandard techniques [Hoogenraad and Wraight, Methods Enzvmol. 121 375-381, (1986)] and antibodies were purified by the caprylic acid precipitation method aε described [Reik et al . , J. Immunol. Methods 100. 123 (1987)] . Mabs BBA 1 and BBA 2 were purchased from British Biotechnology (Oxford, England) while Mab ENA-1 was purchased from San Bio (Uden, the Netherlands) Adhesion assays Confluent cultures of HUVECs plated onto gelatin-coated 96 well tissue culture plates (Costar Corp., Cambridge, MA) were treated for 4 hrs. with rhIL-la (550 pg/ml) and rhTNF (400 U/ml) . Wells were waεhed three ti eε with DPBS and incubated for 1 hr. at 37°C in DPBS containing 1% BSA and 10 ug/ml of designated MAb. HL60 cells were washed twice and resuεpended in RPMI Medium 1640 (GIBCO Laboratories Grand Island, N.Y.) at 5 X 10 6 cells/ml and labeled for 30 min. at 37°C with 40 ug/ml 6 Carboxyfluorescein (6-CFDA) . 6-CFDA loaded HL60 cells (100,000/well) were added and incubated for 20 min. at 25°C. Wells were filled with RPMI and plates were sealed, inverted and spun for 6 min. at 500 X G. Non-adherent cells were removed by aspiration and plates read in a CytoFluor 2300 fluorescent plate reader (Millipore Corp., Bedford, MA.) . COS cells were plated at 5 X 10 J cellε/35 mm-diameter well 18 hr prior to transfection. Cells were washed with DPBS and 2 μg DNA was added in 1 ml DMEM containing 10% Nutridoma (Boehringer-Mannheim, Indianapolis) , 50 μM chloroquine, and 500 ng/ml DEAE dextran. After incubation for 2.5 hr at 37°C, the wells were aspirated and the cells were incubated in Iscove's modified Dulbecco's medium (IMDM) containing 10% FBS and 10% DMSO for 2-5 min. Wells were aspirated and cells grown in IMDM containing 10%FBS at 37°C for 48-72 hrs. For adhesion assayε, 5 X 10 6 6-CFDA loaded HL60 cellε were added to each 35mm diameter well and incubated for 30 min. at 25°C. Wellε were washed 3 times with RPMI and the fluoreεcence associated with adherent cells determined in the CytoFluor 2300 plate reader. Indirect immunofluorescencβ Transiently transfected COS cellε were fixed in DPBS containing 1% (wt/vol) formaldehyde for 15 min at 25°C. Following two washes with DPBS, the cells were blocked with DPBS containing 10% horεe εerum (DPBS/10%HS) for 30 minuteε at room temperature. Cellε were incubated for 30 min with 5 ug/ml mAb'ε 387, 8E4, 7H5, or 14G2 in DPBS/10%HS and then washed three times with DPBS. Following a 30 min incubation with rhodamine-conjugated goat anti-mouse IgG, cells were washed with DPBS, and fluorescence observed on a Zeiss Axioεkop microscope. Human-rabbit chimeric E-Selectin constructs Expreεsion of truncated forms of human and rabbit E-Selectin on the surface ' of COS cells was achieved by fusing the selectin sequences with the carboxy terminal 37 amino acids of CD16 which contains the εignal εequence for cell εurface anchorage via a glycosyl-phosphatidylinoεitol linkage (GPI) [Scallon e al., Proc. Natl- Acad. Sci- USA 86, 5079-5083 (1989)] . All E-Selectin fragments were generated by polymerase chain reaction (PCR) and cloned into the plasmid vector pBC12BI [Cullen,B.R. Methods in Enzvmology (eds. Berger, S.L., and A.R. Kimmel) 152, 684-704 (1987)] * that had been modified to contain the CD16 εequenceε. Recombinant geneε were expreεεed using the initiating Met codon from the rat preproinsulin gene in pBC12BI and the primary tranεlation product contains five amino acids derived from the insulin εignal εequence. The human lectin-egf construct contained amino acids -15 through +157 [Bevilacqua et al., Science 243. 1160-1165 (1989)], the rabbit lectin-egf contained residues -17 through +156 [Larigan et al., J. of DNA and Cell Biology " 11, 149-162 (1992)], while the HuRa-l contained amino acids -15 to +9 of human E-Selectin contiguous with residueε 10 through 156 of rabbit E-Selectin. The CD16 sequences required for GPI anchorage to the cell surface were fused to the carboxy termini of each construct. Construction and expression of E-Selectin-IgG chimera mutants Production and characterization of the E-Selecti -IgG chimera has been previously described (Foxall et al . , 1992, Supra) . Mutations were introduced into the lectm domain of this chimera by the method of Kunkel [Kunkel et al., Methods in Enzvmol. 154. 367-382 (1987)] using the Muta- Gene Phagemid in vitro Mutagenesiε Kit (BioRad) according to the manufacturer'ε inεtructionε. Correct mutants were confirmed by DNA sequencing and the mutant chimeraε were transiently expressed and secreted by transfection of 293 cells [Watson et al . , Nature 349, 164-167 (1991)] . The concentration of each chimera in the resultant εupernatants were then quantified by ELISA using an anti-human IgGl-Fc specific mouse mAb as previously described [Watεon et al . , J. Cell Biol. 110. 221-2229 (1990)] . Point mutation εiteε are defined using the nomenclature of Cunningham and Wells (1989) : wild type residue-position-mutant residue. Oligonucleotide sequences used for mutagenesis: SEP. NAME SEQUENCE (5'-->3') ID. No. E Selectin-E8A 5' -ATA AGT CAT AGC GGC CGT GGA GGT GTT-3' 1 E Selectin-MIOA 5'-CTC ATT ATA AGT GGC AGC TTC CGT GGA-3' 2 E Selectin Y12A 5'-ACT GGC CTC ATT GGC AGT CAT AGC TTC-3' 3 E Selectin E14A 5' -ATA AGC ACT GGC TGC ATT ATA AGT CAT-3' 4 E Selectin K32A 5' -CTC AAT CTC TTC TGC GTT TTG AAT TGC-3' 5 E Selectin-S43A 5'-ACT TGG TGA ATA GGC CAA TAT GGA GTT'3' 6 E Selectin- 44A 5' -ATA ACT TGG TGA AGC GCT CAA TAT GGA-3' 7 E Selectin-S45A 5'-GTA ATA ACT TGG TGC ATA GCT CAA TAT-3' 8 E Selectin-P46A 5' -CCA GTA ATA ACT TGC TGA ATA GCT CAA-3' 9 E Selectin-S47A 5' -AAT CCA GTA ATA AGC TGG TGA ATA GCT-3' 10 E Selectin-Y48A 5'-TCC AAT CCA GTA AGC ACT TGG TGA ATA-3' 11 E Selectin-Y49A 5' -GAT TCC AAT CCA GGC ATA ACT TGG TGA-3' 12 E Selectin-K67A 5' -TTC TTC TGT CAG AGG CGC CTG GGT TCC 13 TAC CCA-3' E Selectin-K74A 5'-GGG TTC ACC TGG CGC CCA GTT CGC GGC 14 TTC TTC TGT-3' E Selectin- 5'-CAC GCA GTC CTC ATC TGC TTG CGC ATT 15 R84A,K86A GTT GGG TTC ACC-3' E Selectin-E92A 5'-CTT GAT GTA GAT GGC CAC GCA GTC CTC-3' 16 E Selectin-I93A 5'-TCT CTT GAT GTA GGC CTC CAC GCA GTC-3' 17 E Selectin-Y94A 5'-TTC TCT CTT GAT GGC GAT CTC CAC GCA-3' 18 E Selectin-195A 5' -TTT TTC TCT CTT GGC GTA GAT CTC CAC-3' 19 E Selectin-K96A 5'-ATC TTT TTC TCT CGC GAT GTA GAT CTC-3' 20 E Seleetin-R97A 5' -CAC ATC TTT TTC AGC CTT GAT GTA GAT-3' 21 E Selectin-E98A 5'-GCC CAC ATC TTT GGC TCT CTT GAT GTA-3' 22 E Selectin-K99A 5' -CAT GCC CAC ATC GGC TTC TCT CTT GAT-3' 23 E Selectin-D100A 5' -CCA CAT GCC CAC GGC TTT TTC TCT CTT-3' 24 E Selectin-VIOIA 5 r -ATT CCA CAT GCC AGC ATC TTT TTC TCT-3' 25 E Selectin-M103A 5' -CTC ATC ATT CCA TGC GCC CAC ATC TTT-3' 26 E Selectin-E107A 5' -CTT GCT GCA CCT CGC ATC ATT CCA CAT-3' 27 E Selectin-K111A 5' -GGC AAG CTT CTT GGC GCT GCA CCT CTC-3' 28 E Selectin-K113A 5 r -GCA TAG GGC AAG AGC CTT CTT GCT GCA-3' 29 E Selectin- 5' -AGC TTC CGT GGA GTA GTG GTA AGT CCA 30 S2T,N4H,T5Y GGC TCC ACT-3' E Selectin-T7A,A9N 5' -ATT ATA AGT CAT ATT TTC CGC GGA GGT 31 GTT GTA-3' E Selectin- 5' -ACT GGC CTC ATT CCA ACT GTA AGC TTC 32 M10Y,T11S,Y12W CGT GGA-3' E Selectin- 5' -ATT CCA CAT GCC CGT ATC TTT TGG TCT 33 E98P,V101T CTT GAT GTA-3' L Selectin-K8A 5' -CCA GTT CAT GGG GGC TTC AGA ATA ATG-3' 34 P Selectin-K8A 5'-CCA TGA GTA TGC AGC TGT GCT GTA ATG-3' 35 Monoclonal antibody binding of E-Sβlβctin-IgG mutants Reactivity of the mutant E-Selectin-IgG chimeras with the various antibodies was determined using a previously described ELISA format [Watson et al. , 1990, Supra) in which the purified Mab were coated onto microtitre wells, then blocked with BSA.293 cell supernatantε containing equal concentrations of wild type or mutant chimeras were incubated in the wellε, followed by washing and detection of the captured chimeras with HRP-conjugated goat polyclonal anti-human Fc antibody. Sialyl Lewis X binding of E-Selecti -IgG mutants Aεεays for binding of the mutant E-Selectin-IgG chimeras to immobilized sLex glycolipidε were performed esεentially aε described (Foxall et al., 1992, Supra) . Briefly, sLex glycolipidε were drie .onto microtitre wells, washed with distilled water, and then blocked with BSA. Biotinylated goat F(ab') anti-human IgG Fc and alkaline phosphataεe- εtreptavidin (Caltag, South San Franciεco, CA) were each diluted 1:1000 into 293 cell supematants containing equal concentrations of wild type or mutant chimeras and allowed to form a complex prior to addition to the wells. These supematants were then incubated on the sLex glycolipid coated εurfaces, followed by washing, addition of subεtrate (p- nitrophenyl phoεphate) , and meaεurement of the O.D. at ' 405 nm. Generation of a Model of the E-Selectin lectin domain The model of E-Selectin waε generated baεed on the cryεtal structure of the rat mannose-binding protein (MBP) [Weis et al. , 1991, Supra) . The sequence of E-Selectin waε aligned with thoεe of mouse L-Selectin (LHR) [Lasky et al., Cell 56. 1045-1055 (1989)] and MBP using the alignment of the latter two proteins provided [Weis et al . , 1991, Supra) . Eleven insertions and two deletions in E-Selectin relative to MBP mapped to four εurface loopε in the MBP εtructure. MBP (molecule 1) waε tranεformed into E-Selectin in three steps. First, all residueε except thoεe involving insertions/deletions were changed to the E-Selectin sequence using the INSIGHT-II program (Biosym Technologieε, San Diego) . If possible, conformations of E-Selectin side chains were kept similar to those of MBP, otherwise they were baεed on rotamer libraries [Ponder and Richards, J. Mol. Biol. 193. 775-791 (1987)], packing and hydrogen- bonding considerations. Second, possible loop structures for the E- Selectin inεertionε/deletions were gleaned from a search of crystal structures in the Protein Data Bank [Berstein et al., J. Mol. Biol. 112. 535-542 (1977)] using the INSIGHT-II program. Third, each of the thirty water molecules present in the MBP crystal εtructure was evaluated regarding its retention in the E-Selectin model. Only seven waters were included in the E-Selectin model, four of which corresponded to MBP water molecules 23, 24, 25 and 30. The E-Selectin model was subjected to 6000 cycles of energy minimization using the DISCOVER program (Biosym Technologies, San Diego) . The all-atom AMBER forcefield [Weiner et al. J. Am. Chem. Soc. 106. 765- 784 (1984), Weiner et al . J. Comp. Chem. 2, 230-252 (1986)] was used for all calculations, employing a 14A cutoff for nonbonded interactions and a linear dielectric (e=4.0*r). Hydrogen atoms were added to the εtructure using INSIGHT-II and positionε of hydrogenε on Ser, Thr and Tyr εide chainε and on water moleculeε were checked visually for proper alignment in hydrogen-bonds, if present. Energy minimization waε performed in six stageε of 1000 cycles each. In stage l, steepeεt- deεcentε minimization waε employed with Ca atomε of reεidueε S2-V27, Q30- S40, Y49-R54, W60-V61, N75-N82, E88-I93 and W103-T119 constrained to their initial positions using a force constant of 100 kcal/A. 105 hydrogen-bond constraints (50 kcal/mole) were also invoked involving primarilyhydrogen-bondε between reεidueε in the b-εtrands and a-helices. Thiε allowed loop structures and εidechainε to move while preεerving the integrity of the secondary structure present in the E Selectin model. In stages 2 and 3, the Ca tether force constant was reduced to 50 and 10 kcal/A, respectively, and conjugate gradients minimization waε utilized. In εtageε 4 and 5, the Ca constraints were released and, finally, in stage 6 the hydrogen-bond conεtraintε were released. The MBP crystal structure includes two Ho 2+ atoms which occupy the two Ca 2+ -binding sites in MBP. E-Selectin retains one Ca 2+ -binding site, but looses the second (see results) . Since the AMBER forcefield (Weiner et al . 1984, and 1986, Supra) does not include a representation for Ca 2+ , the Ca 2+ atom was removed and the εidechains which coordinate the Ca 2+ (E80, N82, E88, N105, D106) were fixed throughout the minimization procedure. B. Results Characterization of a Panel of E-Selectin Antibodies To facilitate the study of E-Selectin structure and function, we generated a panel of blocking and non-blocking Mabs directed against human and rabbit E-Selectin (see Experimental Procedures) . Three anti- human E-Selectin Mabs (7H5, 8E4, and 3B7) were found to inhibit the adhesion of HL60 cells to cytokine activated HUVECε and E-Selectin transfected COS cellε (Figure 1A) . Cross reactivity studies demonstrated that these three blocking Mabs did not recognize rabbit E-Selectin (Figure IB) , a result that facilitated the mapping of the epitopes recognized by these Mabs (see below) . The commercially available anti- human E-Selectin MAbε, BBA1, BBA2 and ENA-1, alεo did not croεs react with rabbit E-Selectin (Figure IB) . While none of these three commercial MAb's significantly blocked E-Selectin-mediated HL60 adherence in our cell adhesion assay (Figure 1A) , BBA2 has clear adhesion blocking activity in cell adhesion assays done at low temperature [Pigott et al., J. Immunol- 147. 130-135 (1991) ,- C. Phipps-perεonal communication] and ENA-1 has been shown to block neutrophil adhesion to activated HUVECs [Leeuwenberg et al., Clin. exp. Immunol. 81496-500 (1990)] . Furthermore, BBA2 and ENA-1 both effectively inhibit binding of E-Selectin to immobilized εLex glycolipid (Foxall et al., 1992, Supra) . Since εLex is the major carbohydrate ligand for E-Selectin on the leukocyte cell surface, it seemed likely that analysis of the epitopes recognized by this panel of blocking antibodies (7H5, 8E4 , 3B7, BBA2 and ENA-1) would indicate the region(ε) of E-Selectin involved with carbohydrate recognition and resultant cell adhesion. In addition, the mapping of regions recognized by other, non-blocking Mabs should confirm and emphasize the site(ε) found for blocking antibodieε by indicating regions of the lectin domain not involved with carbohydrate recognition and cell adhesion. Therefore, the initial step in analyzing the regions of E- Selectin involved in carbohydrate interactions consisted of mapping the epitopes recognized by blocking and non-blocking anti-E Selectin Mabs. Analysis of E-Selectin Monoclonal Antibody Binding The E-Selectin mutagenesis strategy was driven by two major considerations. The first consideration derived from previous work on the localization of the epitope recognized by the murine L-Selectin blocking Mab, Mel 14 [Bowen et al., J. Cell Biol. 107. 1853-1862 (1990)] . This work demonstrated that thiε antibody recognized a region within the N- terminal 53 amino acids of murine L-Selectin. It waε assumed that blocking antibodies directed againεt E-Selectin may alεo recognize epitopeε contained within the N-terminuε of the lectin domain, and this region waε, therefore, targeted for mutagenesis. A second strategy targeted positively-charged reεidueε in the lectin domain uεing both alanine-scanning and species homologue mutagenesis protocols [Cunningham and Wells, Science 244. 1081-1085 (1989)] . The fact that all selectinε require a.negatively charged sialic acid as part of the carbohydrate ligand along with other observations could be interpreted aε indicating the formation of a charge-mediated interaction between the negatively- charged carboxylate of the sialic acid and one or more positively charged residueε in the lectin domain. As'described above, none of the anti-human E-Selectin blocking Mabs reacted with rabbit E-Selectin, and analysis of the amino acid sequences of the lectin domains of human and rabbit E-Selectin showed that 5 of 16 differenceε were clustered in the N terminal nine amino acids (Figure 2A) . To determine whether blocking MAbs map to thiε region, a chimeric protein containing rabbit E-Selectin lectin and egf-like domainε with the N-terminal 9 amino acidε replaced by the correεponding sequence from human E-Selectin was generated (Hu-Ra-1) (see Figure 2B) . This construct was produced as a fusion of the lectin and egf-like domains with a region of CD16 εufficient to allow anchoring of the expressed protein to the cell surface by a glycosyl-inoεitol phosphate linkage (Figure 2B) . The egf-like domain was included because previouε data εuggeεted that the overall conformation of εelectin lectin domainε required the preεence of the adjacent egf-like domain. Indirect immunofluorescence on COS cells transfected with human lectin-egf-CD16, rabbit lectin-eg -CD16, or the human-rabbit chimera (Hu-Ra-1) demonεtrated that human amino acids 1-9 in the rabbit E-Selectin background were sufficient to confer MAb 7H5 and 8E4 binding but not MAb 3B7 binding (Figure 3) . In similar experiments, ENA-1, but not BBA2, bound to HuRa-l (not shown) . In addition, the adhesion of HL60 cells to Hu-Ra-1 transfected COS cells waε inhibited by MAb's 7H5 and 8E4, but was unaffected by MAb 3B7 or the non-blocking MAb 14G2 (Figure 4) . These data were consistent with the localization of the epitopes recognized by three blocking Mabs (7H5, 8E4 and ENA-1) to the N-terminal 9 amino acids of human E-Selectin. To facilitate further Mab mapping analyεis and allow for direct carbohydrate binding studies (see below) , mutations were introduced into the lectin domain of an E-Selectin-human immunoglobulin G (IgG) chimera that iε εimilar to a previouεly deεcribed L-Selectin-IgG chimera (Figure 2B) [Watson e l., 1990, 1991, Supra.- Foxall et al., 1992, Supra) . The E-Selectin-IgG chimera allowed for easy uantitation of each individual mutant by analyεiε of the amount of human IgG produced from each transient cell transfection assay (see Experimental Procedures) . The inclusion of the human IgG tail also allowed for rapid analysis of the ability of each mutant to bind the anti-E-Selectin antibody panel, as well aε to immobilized εLex, by uεe of labeled anti-IgG antibody. In this way, mutants that affected global lectin structure (losε of recognition by all MAbε) , localized εtructure (losε of recognition by a subset of Mabε) , and carbohydrate recognition (losε of sugar recognition with retention of recognition by most or all Mabε) could be rapidly differentiated. Figure 5 εhowε a number of mutationε that appear to affect the binding of variouε monoclonal antibodieε to E-Selectin- Since the chimeric construct of the lectin domains of human and rabbit E-Selectin (HuRa-l) identified the N-terminal nine amino acids aε forming at least part of the epitopes for three blocking antibodieε (8E4, 7H5 and ENA-l) , we first constructed further mutants within this region, concentrating on the five poεitionε (reεidueε 2, 4, 5, 7, and 9) which differ between rabbit and human. A human E-Selectin-IgG chimera in which reεidueε 2, 4, and 5 were mutated retained binding to the entire panel of aπtibodieε, indicating that these amino acidε are not critical for MAb binding (data not εhowπ) . However, a mutation which replaces the human E-Selectin amino acids at poεitionε 7 and 9 with their counterparts found in the rabbit E-Selectin sequence resulted in losε of binding of antibσdieε 7H5, 8E4 and ENA-l (Figure 5A) . The loεε of binding of theεe 3 mAb'ε directly corresponds to the gain of binding demonstrated with the HuRa-l chimera. Another N-terminal mutant, E8A, was found to abolish the binding of BBA2 and ENA 1 (Figure 5C) . Thus, in agreement with the human-rabbit chimera studies described above, residues at positions 7, 8, and 9 of the E-Selectin lectin domain contribute to the epitope recognized by four anti-E-Selectin blocking Mabs. The mutations at positionε 7, 8 and 9 enabled the mapping of all blocking antibodies except for Mab 3B7. Replacement of the human residues with their rabbit counterparts at other siteε of the E-Selectin lectin domain revealed that a double mutation at reεidueε 98 and 101 completely abolished the binding of this blocking Mab (Figure 5B) . Importantly, the converse experiment was also done where the human residues at these two sites (98 and 101) were placed into the HuRa-l background. The resultant mutant was found to bind Mab 3B7, thus confirming that this site contained an epitope recognized by this blocking Mab (data not εhown) . Finally, an E-Selectin-IgG mutant which contained a valine to alanine replacement at poεition 101 retained binding to Mab 3B7, indicating that the E at position 98 was a crucial component of the 3B7 epitope. The complexity of the epitope recognized by the 7H5 blocking Mab is revealed by the mutation K113A (Figure 5C) . This mutation, which is at the C-terminus of the lectin domain, completely abolishes binding of this MAb. Since the binding of 7H5 was also abolished by two point mutations made at the N-terminus of the lectin domain (positionε ' 7 and 9, Figure 5A) , it may be concluded that the epitope recognized by thiε blocking antibody iε derived from both the N- and C-termini. Indeed, the partial loss of 7H5 binding found for mutationε at residues 98 and 101 (Figure 5B) was consistent with the close alignment of this region with the N and C terminal sites recognized by this Mab as well. One interpretation of this result is that these regions may be cloεely aligned in the tertiary εtructure of the lectin domain (εee below) . The binding of all of the non-blocking Mabε did not appear to be grosεly affected by mutationε that diεrupted blocking Mab recognition (Figureε 5A-D) . This was consiεtent with these two clasεeε of Mabs recognizing distant sites in the lectin domain. For example, mutant K74A completely lost binding of a number of these non-blocking antibodies (9H9, 1B3, 11G5, 4D9 and 1D6) and partially lost binding of another (14G2) (Figure 5E) . Thiε mutant did not affect the binding of any of the blocking Mabs that we analyzed, suggesting that this region of E-Selectin may not be directly involved in carbohydrate recognition. Lastly, a number of mutants (M10A, Y12A, E14A, I93A, Y94A, I95A, K96A, E98A and M103A) resulted in the loss of binding to all of the antibodies in the anti-E-Selectin panel, including 9A1 and 7E10 which recognize determinants in the complement binding like domains 1 and 2 of E-Selectin. This result was obtained even though normalized amounts of these mutants, based upon human IgG concentrations, were added to the antibody-coated wells. These mutations, therefore, appeared to have global effects on the recombinant E-Selectin domains, and the apparent lack of monoclonal antibody reactivity could have been due to denaturation and/or degradation. Carbohydrate Recognition by E Selectin Mutants While the blocking Mab mapping data deεcribed above were consistent with the involvement of the N- and C-terminal regions as well as the region surrounding residueε 98 and 101 of E Selectin with carbohydrate recognition, the large shadow cast on an antigen by a bound monoclonal antibody [typically 680-880 A 2 [Jin et al . , 1992, Supra; Davies and Padlan, Ann. Rev. Biochem. 59, 439-473 (1990)] may cause blocking by steric hindrance of carbohydrate recognition siteε relatively distant from the antibody epitope. Therefore, we analyzed the ability of a number of the E-Selecti -IgG mutants to bind to sLex glycolipid [Kameyama et al.. " Carbohydrate Res. 209. cl-c4 (1991)] that had been immobilized on plastic microtitre wells. Thiε aεεay haε been validated previously (Foxall et al., 1992, Supra) . and the binding of the E-Selectin-IgG chimera has been shown to be calcium dependant, inhibited by E-Selectin blocking Mabs and dependant upon the presence of the alpha 2-3 sialic acid form of the carbohydrate. As can be seen in Figure 6A, three different types of resultε were obtained with the various mutants analyzed. A number of mutations did not affect the binding of E Selectin to immobilized sLex. Theεe mutationε were found to be of the type that either did not affect the binding of any monoclonal antibody (mutantε K32A, K67A, and R84A, K86A) or affected binding of only non-blocking antibodieε (mutant K74A) . This result was consistent with the previously mentioned possibility that this region of E-Selectin waε not involved with carbohydrate recognition- Another " type of effect waε exemplified by mutationε at positions R97, K99 and K113. Conversion of any of these siteε to alanine either completely (R97 and K113) or almost completely (K99) aboliεhed the binding of E- Selectin to sLex. While mutations at R97 and K99 were found to have no effect on the binding of blocking antibodies, mutation at K113 was found to completely abolish the binding of blocking antibody 7H5 (εee above) . In addition, mutation at sites 98 and 101, adjacent to R97 and K99, aboliεhed the binding of another blocking Mab, 3B7. Theεe reεultε unified the locations of blocking Mab binding sites with residueε that were critical for carbohydrate recognition, and were consistent with the direct involvement of these regions of E-Selectin with carbohydrate binding. The final, somewhat unexpected, effect of E-Selectin mutation on εLex binding waε exemplified by the E8A mutant. Figure 6A εhowε that this mutant appeared to show enhanced binding to εLex when the mutant waε added to wells at the same concentration as wild type E-Selectin chimera. A dose reεponεe curve comparing the E8A mutant with wild type E-Selectin (Figure 6B) revealε that the mutant bound -5 fold more avidly to sLex than wild type. This enhanced binding by mutant E8A waε completely calcium dependant and did not occur on the inactive alpha 2-6 sialic acid form of the carbohydrate (data not εhown) . Aε described previously, mutations in this region (i.e. at residueε 7,8 and 9) profoundly affect the binding of εeveral blocking Mabε. Thus, it iε likely that this region of E-Selectin alεo plays a critical role in carbohydrate recognition. A Model of E Selectin While the resultε deεcribed above allow for a number of important concluεions about the regions of the E-Selectin linear sequence involved in carbohydrate recognition, their relevance would be enhanced if they could be applied to a structural model of the lectin domain of this protein. In the absence of crystallographic data, a three-dimensional model of a molecule can be constructed using structural coordinates of related molecules [Greer, J. Mol. Biol. 153. 1027-1042 (1981)] . Recently, the X-ray structure of the mannoεe binding protein (MBP) , a type C lectin that is homologous to the lectin domain of E-Selectin, was determined (Weis et al . , 1991, Supra) . In order to more fully underεtand the relative importance of the functional mutations described above, we, therefore, used these structural data to develop a model of the E- Selectin lectin domain. Derivation of the E-Selectin lectin model from the MBP coordinates was based on secondary structure common to both, encσmpasεing 78 (of 121) E-Selectin reεidues (Weis et al . , 1991, Supra) (Figure 7) . Three models of E-Selectin were evaluated, differing only in the conformation of two surface loopε: S43-Y48 and Y94-D100, correεponding to MBP K152-S154 and V199-D200, respectively. The S43-Y48 loop contains a three residue insertion relative to MBP while the Y94-D100 loop contains a five residue insertion.. The E-Selectin model prior to energy-minimization had a root- mean-square (r.m.s.) deviation of 0.17A (78 Ca atoms) while that of the best energy-minimized model waε 0.68J For comparative purposes, we also subjected the MBP crystal structure to the same energy-minimization regimen- as utilized for the E-Selectin model. The minimized MBP (molecule 1 only) showed a Ca r.m.s. deviation of 0.45A (residues K110- C217, i.e. excluding seven N- and C-termini residues) versus the crystal structure. Hence the energy-minimization regimen maintained th secondary structure of the E-Selectin model; thiε was accomplished b initially constraining the secondary εtructure Ca atoms and hydrogen- bonds during minimization. Weis (Weis et al . , 1991, Supra) noted the preεence of εmall an large hydrophobic coreε in ?J3P that were critical for overall εtructure. For the εmall core only two of εix residues are conserved in E-Selecti (G52, A115) though the model could accommodate the other four E-Selecti side chains without disturbing the protein fold. In the large core onl six of fourteen residues are conserved. One substitution, A155 (MBP) t Y49 (E-Selectin) , necessitated moving a-helix-2 (K32-L42) slightly awa from the protein center in order to accommodate introduction of the Y4 sidechain. Thiε Y49 εide chain interactε with the two loopε S43-Y48 an Y94-D100 mentioned above. At the oppoεite (N-terminuε) end of the helix the εubstitution of P138(MBP) to 129(E Selectin) also contributed to th slight shift in a-helix-2. However, the substitutions in E-Selectin i the large hydrophobic core fill the internal space created by the sligh shift of a-helix-2. Thus, the relatively large number of amino aci changes-in the hydrophobic core regions of MBP and the E-Selectin lecti domain could be accommodated by the model. The MBP crystal structure contains two putative Ca 2+ -binding site which, in the crystal structure, are occupied by two Ho :+ ions. As note by Weis, Supra, site 2 is retained in E-Selectin: E80 Oe2, N82 Odl, E88 Oe2, N105 Odl, D106 Od2, D106 backbone carbonyl oxygen, and one water molecule coordinate this Ca 2+ . The other Ca 2+ -binding site is probably not present in E-Selectin, alεo noted previouεly (Figure 7) - While D89 (D194 in MBP) is conserved in E-Selectin, D161 in MBP is replaced by K55 in E-Selectin and two other side chainε which coordinate the MBP site 1 Ca 2+ , i.e. E165 and D188, are replaced by N57 and N83 in E-Selectin. While an Asn side chain could still coordinate a Ca 2+ via its Odl atom, N57 iε adjacent to a two residue deletion and the loop of which it is a part probably changes conformation. Likewise, N83 is part of a loop which has a proposed conformation in E-Selectin different from that in MBP. Though this loop containε no insertions or deletions, the MBP sequence HGSG forms a Type II' reverse turn with the Gly at position 4 having a backbone conformation allowable only for Gly (136°, 148°) . The E-Selectin εequence RQKD neceεsitated a different loop conformation due to the G to Q and G to D replace entε. Thuε, only one calcium binding site appears to exist in the E-Selectin lectin domain. Of.course, the moεt interesting aspect of the model is the location of the amino acid side chainε whoεe mutation appeared to affect monoclonal antibody binding and/or sLex recognition. As can be seen from the model (Figure 7) , the amino acid residues involved in blocking Mab recognition appear to form a patch on the surface of the lectin domain near the antiparallel beta sheet formed by the disulfide-linked N- and C-termini and the adjacent disulfide-linked loop formed by the two internal cyεteineε. Thuε, amino acid side chains at positions 7,8,9, 98, 101 and 113, all of which affected the binding of various blocking Mabs, are all found to be very close to each other on the surface of E-Selectin (within a patch of -80 square angεtromε) . Of particular importance was the close alignment of the side chainε of residueε 7, 9 and 113 which waε conεiεtent with the effect of mutationε at theεe reεidueε on the binding of the 7H5 blocking Mab. The partial effect of mutationε at reεidueε 98 and 101. on 7H5 binding is also consistent with the model, since the diεulfide-bonded loop containing theεe reεidueε is also close to residueε 7, 8, 9 and 113. The model alεo reveals that the side chain recognized by the non-blocking antibodies (K74) iε found on a side of the lectin domain that iε oppoεite to that which appearε to bind the blocking antibodieε (Figure 7) . Thiε result iε conεistent with the region recognized by non-blocking antibodieε being a considerable distance from the carbohydrate recognition site. Thus, the model of E-Selectin suggests that a relatively εmall patch of the molecule recognized by blocking monoclonal antibodies appears to be involved with carbohydrate recognition. Examination of the location amino acids found to be involved with sLex binding is consistent with the interpretation that the region recognized by the blocking monoclonal antibodies is involved with carbohydrate recognition. The close spacing of residue E8, whose mutation was found to enhance εLex binding, and residue K113, whose mutation was found to abolish carbohydrate binding, can be clearly seen on the model. Perhaps more interesting is the close proximity of two other mutations that either completely or almost completely abolish sLex recognition. Aε can be εeen from the model in Figure 7, residue R97, whose mutation virtually abolished εLex recognition, is found quite close to the K113 εite whoεe mutation alεo aboliεhed εLex binding. The K99 mutation, which alεo showed a profound negative effect on sLex binding, was also found near this region. Mutation of another residue within this same loop (D100) had no effect on εLex binding (Figure 6A) . Theεe reεults are consistent with the recognition of sLex by a relatively small region of the E-Selectin lectin domain comprised of residueε from the N and C termini and the εmall disulfide-bonded loop. In addition, they also suggest a role for at least one positively-charged amino acid side chain (R97, K99 or K113) in the recognition of this carbohydrate ligand. Finally, the model εhown in Figure 7 demonεtrateε that a number of εide chaiπε whoεe mutation did not affect sLex binding are located on a side of the molecule that is quite distant from the apparent sLex binding εite. C. Discussion The foregoing data provide valuable information about the interactions between E-Selectin and its carbohydrate ligand, sLex. The work disclosed in thiε example iε unique in that it combineε the mappin of blocking and non-blocking monoclonal antibody binding εiteε with the ability of a given E-Selectin mutant to bind to itε natural carbohydrat ligand, εLex. The agreement between blocking Mab mapping data and th location of mutationε that affect εLex binding iε consistent with th hypothesiε that the small region of the E-Selectin lectin domai identified here is directly involved with binding to the previously- defined face of sLex (Tyrrell et al. , 1991, Supra; Berg et al. , 1991, Supra) . This proposal is also supported by the fact that many non- blocking monoclonal antibodieε appear to map to the σppoεite εide of th lectin domain. One of the most interesting aspectε of the mutagenesis describe here is .the discovery that two positively charged residueε (R97 and K113) that are critical for sLex recognition appear very close together on th E-Selectin model (figure 7) . A major supposition of the mutageni analysiε waε baεed upon the potential importance of poεitively charge residues, and the discovery that the deletion of either of these sid chains has negative effects on carbohydrate recognition supports th possibility that positively charged side chains in Selectin lecti domains.are involved in carbohydrate recognition (Stoolman et al . , 1987 Supra.- Yednock et al., 1987, Supra; Lasky et al., 1989) . One poεεibl means by which this recognition could be accomplished is by charge interaction between these side chainε and the carboxylate group of the εialic acid reεidue found in sLex. The importance of the carboxylate group in εLex binding iε underlined by organic synthesis studies that have revealed that an sLex-like compound with only a carboxylic acid group at the εialic acid site can be effectively recognized by E Selectin (J. Musser and B. Brandley-unpubliεhed obεervations) . Thus, it seems likely that some type of charge-mediated interaction may be involved with sLex adhesion to E Selectin. All three εelectinε appear to require sialic acid for adhesion, and it iε intereεting that, while K113 iε found in all three εelectins from a number of different species, a positively charged residue (R) is found at position 97 of L- and E-Selectin only, while human P-Selectin contains an S at this site. The conservation of a K at position 113 of all εelectins, together with the mutageneεiε analyεiε deεcribed here, iε conεistent with a direct role for thiε residue in sialic acid recognition, perhaps by the formation of a εalt bridge or hydrogen bond. The less stringent conservation between selectins at position 97 argues that thiε residue, while clearly involved with carbohydrate recognition, may have a lesε direct effect on εugar binding than residue K113. Interestingly, mutation of D100 to alanine showed no effect on sLex binding, consistent with the εuppoεition that only the poεitively charged residues in this area are involved with sLex recognition. The data disclosed here also provide strong evidence for involvement of the N-terminus of E-Selectin in carbohydrate binding. Mutagenesiε of the N-terminuε waε initially inspired by previous data that demonstrated that the anti-murine L-Selectin blocking Mab, Mel 14, appeared to map to the N-terminus of this glycoprotein. In agreement with that study, a number of anti-E-Selectin blocking monoclonal antibodies were found to recognize residueε in the N-terminus of this glycoprotein aε well. In fact, of the five blocking antibodies that we have analyzed, four (BBA 2, ENA 1, 8E4.and 7H5) have been directly shown to bind to thiε region of E-Selectin, conεiεtent with the important role of this site in carbohydrate recognition. The finding of a residue (E8) in this region whose mutation to A enhanced carbohydrate binding was also consistent with the involvement of this site in sLex recognition. In addition, the enhanced recognition of sLex by mutation at position 8 may be of biological εignificance. Aε waε pointed out earlier, selectinε appear to mediate a relatively low affinity "rolling" type of adheεion aε a precursor to firmer adhesion mediated by leukocyte integrins (Butcher 1991, Supra,- Lawrence and Springer 1991. Supra; Ley et al., 1991, Supra; Von Andrian et al., 1991, Supra) . It is poεsible that the N terminal region of E selectin has evolved to decrease the relative affinity of carbohydrate recognition by incorporation of a charged residue at this site. Interestingly, the other two εelectins (L- and P-) contain a poεitive charge at this site (K) , and mutation of the K in L- Selectin to an A also appears to similarly enhance sLex recognition by 5 to 10 fold (εee Example 2 ) . It thuε εeemε poεεible that the charge at this site may serve to decrease the affinity of carbohydrate binding, consistent with a close asεociation of carbohydrate with this region. While the reεidueε delineated here may be directly involved with sLex recognition, indirect effects may alεo play a role in the losε of binding by εome of theεe mutantε. For example, mutation of residue K99 to alanine partially decreased εLex recognition by E-Selectin. Thiε residue was relatively distant from the closely opposed R97A and K113A mutations which profoundly affected sLex binding, consiεtent with the possibility that the K99A mutation may have affected sLex binding by an indirect mechanism. One such indirect effect would be if the K99 to A mutation changed the conformation of thiε loop εuch that R97 no longer formed a stable contact with the carbohydrate. Another indirect effect may involve interactions between the egf-like and lectin domains. Previous data have clearly demonstrated, in both the L- and E-Selectin adhesion moleculeε, that removal of the egf-like domain results in a loss of epitope recognition by antibodies directed againεt the lectin domain (Bowen et al . , 1990, Supra; Walz et al . , 1990, Supra) . Theεe reεults were consistent with the possibility that one role for the egf domain is in the mediation of lectin domain structure, perhaps by intimate contacts between residues in each of these motifs. It is, therefore, possible that loss of binding by one or more of the mutants described here ma have been due to a decreased level of interaction between the lectin and egf-like domainε. However, it muεt be emphaεized that cryεtallograph studies of point mutants in other proteins show structural differences to be highly localized (Jin et al., 1992, Supra; Wells 1991, Supra an references therein) . Additionally, it should be noted that the retentio of binding by the lectin-specific monoclonal antibodieε to mutantε R97 and K99A is not conεiεtent with the ma^or diεruption of lectin domai εtructure that might be expected by inhibition of εome of th interactionε between the lectin and egf-like domainε (Bowen et al., 1990, Supra; Walz et al., 1990, Supra) . Recently, Geng and coworkerε showed that a mAb capable of inhibitin neutrophil binding to P-Selectin mapped to residues 19-34 of thi molecule, and that a peptide corresponding to this stretch also inhibite neutrophil binding to P-Selectin (Geng et al., 1991, Supra) . In th model of E-Selectin presented here, residueε 19-34 form a loop which i on the oppoεite side from the site where εLex appearε to bind. The tw mutantε made within thiε εequence in E-Selectin either did not affec εLex or mAb binding (mutant K32A) or resulted in a misfolded protei (mutantε Y18A, Q20A, R22A) . Thuε, although an extenεive mutationa analysis of this loop was not performed, our data are not consistent wit its direct involvement in carbohydrate recognition by E-Selectin. In addition to the mutationε described above, another subεtitution, N82D, in the E-εelectin lectin domain waε found to dramatically affect εLex recognition. The reεultε of antibody capture and 2'3 sLex binding studies are εhown in Figure 13. Although thiε εubεtitution did not affect the binding of any of the mAb'ε (A) , it completely aboliεhed binding of E-εelectin to 2'3 εLex (B) . The aεparagine at poεition 82 is predicted to participate in the Ca ++ chelation via its Oδl molecule. Removal of the amino group from thiε aεpargine in the N82D εubεtitution would not be predicted, a priori, to diεrupt thiε interaction- However, it waε thought possible that the N82D mutant had lost ligand binding due to a reduced calcium affinity- To test this possibility, the N82D mutant waε tested for 2'3 sLex binding in the presence of added calcium. As seen in Figure 13 (C) , the N82D mutant did not bind sLex even at calcium concentrations aε high aε 40 mM. Therefore, the loεε of binding by this E-selectin mutant is probably not due to a losε of calcium chelation by the lectin domain. Rather, theεe results implicate a role for the terminal amino group of asparagine at position 82 of the E-selectin lectin domain in ligand recognition by E-selectin. Example 2 Murine L-Selectin-IgG chimeras were prepared esεentially aε described in Example 1 for E-Selectin. Lyεine (K) at amino acid position 8 of the L-Selectin was substituted by alanine (A) following the method of Kunkel et al., 1987, Supra. using the Muta-Gene Phagemid in vitro Mutageneεis Kit (BioRad) according to the manufacturer's instructions. The correct mutant waε confirmed by DNA sequencing, and the mutant chimera was transiently expreεεed and εecreted by transfection of human embryonic kidney 293 cellε (Watεon et al., 1991, Supra) . The concentration of the chimera was quantified by ELISA using an anti-human IgGl-Fc specific mouse mAb as described by Watεon et al., 1991, Supra) . The Sialyl Lewis X binding asεay waε performed eεsentially as described in Example 1. The results are shown in Figure 8. Example 3 A. Experimental Procedures Flow cyto etric assay for P-Selectin ligand The interaction of P-Selectin and its cellular ligand waε εtudied uεing a .flow cytometric assay. Cells used in this assay were either HL60 cells (maintained in high glucose Dulbecco's MEM plus 10% Hyclone FBS) or freεh human neutrophils. Human neutrophils were purified from heparinised peripheral blood by a Ficoll-Hypaque gradient to remove mononuclear cellε, followed by treatment with 3% dextran εulfate to remove red blood cellε. The reεulting cellε were >90% neutrophils. Prior to εtaining with P-Selectin-IgG both cell typeε were preincubated in Dulbecco's PBS/1% bovine serum albumin/0.1% sodium azide/1% normal rabbit serum (staining medium) for 30-60 mins on ice. After this initial incubation, lug of P-Selectin-IgG waε added to lOOul aliquots of 10 6 cells and incubated for 30-60 mins on ice. The cells were then washed with staining medium and resuspended in lOOul of staining medium to which was added 2ul of a phycoerythrin-conjugated F(ab') : goat anti-human IgG (Fc εpecific) . The cellε were incubated for 15-30 mins on ice, washed twice with staining medium, and resuspended in 0.5ml of staining medium prior to flow cytometric analysiε on a FACScan (Becton-Dickinson) . To determine that the staining was an interaction of P-Selectin with its ligand, the staining was also done in the presence of lOmM EGTA. To determine the protease senεitivity and the requirement for εialic acid of thiε interaction, HL-60 cellε in D-PBS and 1%BSA were incubated with either trypεin or Arthrobacter or Cloεtridium εialidases at 37°C prior to resuspendiπg in staining medium. To determine the effect of activation on the expresεion of the ligand, human neutrophils were incubated at 37°C with 50 ng/ml phorbol myristate acetate for 10 mins prior to reεuεpending in staining medium. To examine the ability of various carbohydrates to inhibit staining, reagents were added to cells immediately prior to the addition of the P-Selectin chimera and were present until the cells were washed prior to addition of the second stage antibody. A potential complication of this aεεay aroεe from the use of selectin-IgG chimeraε to stain cells (HL60 cellε and neutrophilε) which bear human IgG Fc receptorε (FcgR, Fanger, M.W., Immunol. Today 10: 92-99 (1989)) . Adding rabbit IgG (in the form of normal rabbit εerum) to the assay medium blocked thiε binding in most caseε. However, in some experiments wit human neutrophilε, it waε neceεsary to add murine mAb'ε to human Fcg (Medarex, Inc., Weεt Lebanon, NH) to the aεεay medium to completely bloc this interaction. Anti-selectin monoclonal antibodies The following anti-human P-Selectin monoclonal antibodies wer purchased to characterize the mutant chimeras: mAb'ε AK-6 (CLB-thromb/6) and CRC 81 from BioDesign International (Kennebunkport, ME) , and mAb A 1.2 from Becton Dickinson (San Jose, CA) . The anti-E-Selectin mAb'ε 9A1, 7E10, 3B7, and 9H9 have been described in Example 1. Construction and expression of wild type and mutant chimeras Production and characterization of the P-Selectin-IgG and E Selectin-IgG chimeras has been previously described (Asa, D. et al., 0 Cell Biol. 117: 895-902 (1992)) . The PE-1 chimera waε constructed i two stepε. Firεt, an EcoRI-Xhol fragment encoding the signal peptide lectin domain, and part of the EGF domain of P-Selectin was removed fro a pRK5/P-Selectin-IgG plasmid. pRK5 is discloεed in EP 307,247 publiεhe 15 March 1989. This fragment waε inεerted into a pRK5/E-Selectin-Ig plasmid which had been digested with EcoRI and Bglll to remove the E Selectin signal peptide and most of the E-Selectm lectin domain. Second, the P-Selectin lectin domain was joined in-frame to the E- Selectin EGF domain via oligonucleotide-directed deletional mutagenesis using the method of Kunkel (Kunkel, T.A. et al. , Methods in Enzvmol. 154: 367-382 (1987)) as described in Example 1. The expressed PE-1 construct consiεted of the εignal peptide and lectin domain from P-Selectin, followed by the EGF, CR1 and CR2 domainε of E-Selectin, and the IgGl hinge, CH2 and CH3 domainε common to both the P-Selectin-IgG and E- selectin-IgG constructs. Amino acid substitutions were introduced into the lectin domain of the P-Selectin-IgG chimera as described in Example 1. Wild type and mutant chimeras were expreεsed and secreted by 293 cells, quantified and tested for anti-selectin mAb reactivity also as described in Example 1. Mutant chimeraε are defined using the nomenclature: K113A iε a mutant where the lysine (K) at poεition 113 iε changed to an alanine (A) . Binding of selectin- gG chimeras to sialyl Lewis x and sulfatides Assays for binding of the different selectin-IgG chimeras to immobilized sLe 1 glycolipids or εulfatides were performed aε described (Aεa, D. et al.. Supra) . Briefly, 2'3 sLe x glycolipids, 2'6 sLe x glycolipidε, or bovine brain εulfatides (Sigma, St. Louis, MO) were dried onto microtitre wells, waεhed with distilled water, and then blocked with BSA. Biotinylated goat anti-human IgG Fc and alkaline phosphatase- εtreptavidin (Caltag, South San Franciεco, CA) were each diluted 1:1000 into 293 cell εupernatantε containing equal concentrationε of wild type or mutant chimeras and allowed to form a complex prior to addition to the wellε. Theεe εuperaatants were then incubated on the sLe x glycolipid or εulfatide coated surfaces, followed by washing, addition of subεtrate (p- nitrophenyl phoεphate) , and meaεurement of the O.D. at 405 nm. Generation of a P-Selectin lectin domain model A model of the P-Selectin lectin domain waε generated baεed on the crystal structure of the rat mannose-binding protein (MBP) (Drickamer, K. et al. , Science 254: 1608-1615 (1991)) as previously described for an E-Selectin lectin domain model (Brandley, B.K. et al., Supra) . Briefly, MBP residues were changed to the P-Selectin sequence with the sidechain cσnformationε kept εimilar to thoεe of MBP where poεεible. Otherwiεe εidechain conformationε were based on rotamer libraries (Ponder, J. W. and Richards, F.M., J. Mol. Biol. 193: 775-791 (1987)), packing and hydrogen-bonding considerationε. Possible loop structures for the eleven insertionε and two deletionε in P-Selectin relative to MBP were gleaned from a search of crystal εtructures in the Protein Data Bank (Bernstein, F.C. et al., J. Mol. Biol. 112: 535-542 (1977)). Finally, the P-Selectin model waε subjected to repetitive cycles of energy minimization using the method described for E-Selectin (Brandley, B.K., et al . , Supra) . Results As a starting point for evaluating the residues in P-Selectin responsible for binding ligand, we developed a flow cytometric assay using the P-Selectin-IgG chimera to stain HL60 cells and neutrophils. Whereas E-Selectin-IgG did not bind HL60 cells or neutrophils in thiε asεay, P-Selectin-IgG staining resulted in a strong fluorescence shift for both cell types (Figure 10, A and B) . Thiε binding waε inhibited by EGTA, reflecting the calcium requirement for P-Selectin's interaction with its ligand. Further controlε indicated that thiε assay using the P-Selectin-IgG chimera reflects the publiεhed characteriεtics of P- Selectin/ligand binding. In particular, treatment of HL60 cells with either trypεin or sialidase abolished staining (Figure IOC) . Furthermore, P-Selectin-IgG staining was inhibited by dextran sulfate and mannose-1-phosphate, but not fucoidin or mannose-6-phosphate (Figure IOC) . Following activation of human neutrophilε with PMA, although surf ce. expression of L-Selectin decreased and surface expression of CD11/18 increased, surface expression of the P-Selectin ligand did not change (Figure 10D) . In addition to neutrophils, monocytes and NK/LGL cells were positive when stained with P-Selectin-IgG (data not shown) , which is consiεtent with the expreεsion of the P-Selectin ligand on these cells. As noted above, the E-Selectin-IgG chimera did not bind HL60 cells or neutrophilε in the εoluble FACS aεεay. We exploited this finding t aid in mapping the region of P-Selectin neceεεary for conferring thiε high affinity binding. Since our εtudy with E-Selectin had localized itε ligand binding site to a region within its lectin domain, we sought t determine if the apparent differences in E- and P-Selectin binding coul be attributed to differences in their lectin domains. Consequently, w constructed a chimera (PE-1) which consisted of E-Selectin-IgG with th E-Selectin lectin domain replaced with the lectin domain from P-Selectin. To εee if thiε chimera waε folded correctly, we tested its binding t antibodies specific for the various domainε of E- and P-Selectin. Th PE-1 chimera reacted well with antibodieε to the CR1 and CR2 domainε o E-Selectin (mAb'ε 9A1 and 7E10, Table I) but not with antibodies to th lectin domain of E-Selectin (mAb's 3B7 and 9H9, Table I) . PE-1 bound t the blocking antibody to P-Selectin (De Bruijni-Admiraal, L.G., et al. Blood 0: 134-142 (1992)) (AK-6, Table I), consistent with th localization of the epitope recognized by this mAb to the lectin domai of P-Selectin. By contrast, the nonblocking antibodies to P-Selectin AC 1.2 and CRC 81, did not recognize PE-1 (Table I) . Thiε latter resul is consiεtent with earlier studies which indicated a contribution o residues within the EGF and/or CR domains of P-Selectin in AC 1.2 bindin (Jutila, M.A., et al . , J . Exp. Med. 175: 1565-1573 (1992)) . Thes results are consistent with the PE-l chimera being correctly folded, and indicate that at least part of the epitope recognized by the blocking mAb AK-6 is localized to the lectin domain of P-Selectin. To determine if transferring the P-Selectin lectin domain onto E- Selectin-IgG transferred carbohydrate specificity, we examined binding of PE-l to variouε immobilized glycolipidε. Thiε binding waε compared to that seen with either P-Selectin-IgG or E-Selectin-IgG. Aε εhown in Figure 11, the PE-l chimera appeared to closely mimic P-Selectin-IgG in binding to all three glycolipids tested: 2'3 sLe* (Figure HA), 2'6 sLe 1 (Figure 11B) and εulfatides (Figure 11C) . Therefore, the lectin domain of P-Selectin appears to be sufficient for transferring specificity in binding to these purified glycolipids. We then tested the PE-l chimera for cell εtaining to see if the P- Selectin lectin domain could also confer the high affinity binding to the P-Selectin ligand on HL60 cellε. Aε seen in Figure 12, the PE-l chimera did bind HL60 cells. However, the shift in fluoreεcence εeen with PE-l staining was not as great as that εeen with P-Selectin-IgG (Figure 12) . Therefore, although the lectin domain of P-Selectin did appear to clearly confer HL60 cell staining, some contribution of the EGF and/or CR1 domain of P-Selectin may be required for full, high affinity binding to these cells. Similar results were seen when neutrophils were stained with these three chimeras (data not shown) . The above results using the PE-l chimera indicated that the lectin domain of P-Selectin contained elements responsible for the differences in binding of E- and P-Selectin to immobilized glycolipids and cells. Therefore, we performed mutagenesis of the P-Selectin lectin domain to further localize the reεidueε reεponεible for the interaction of P- Selectin with itε ligand. P-Selectin mutagenesis was focused on those siteε which in our previous study proved to be important for E-Selectin binding to its ligand. This εtrategy was followed for two reasons- First, aε mentioned above, a wealth of experimental evidence exists indicating similarities in recognition of sugars by E- and P-Selectin. Thus, it iε reaεonable to εuppoεe that a εite important for E-Selectin- mediated adhesion would alεo participate in P-Selectin-mediated binding- The εecond reaεon derived from an experimental consideration. In the E- Selectin study we were able to generate an entire panel of antibodies to serve as structural controls for the effects of point mutations on lectin domain εtructure. This allowed the elimination of amino acid substitutions which grossly affected folding of the E-Selectin lectin domain from consideration. In this study, we were limited to just three anti-P-Selectin mAb's (AK-6, AC 1.2, CRC 81), only one of which (AK-6) was clearly shown to bind a determinant in the lectin domain (see above) . To avoid the generation and analysiε of mutantε which do not bind ligand due to a groεs conformational effect rather than a εpecific εide chain substitution, we restricted our analyεiε to only thoεe mutationε which had resulted in correctly folded proteins in the E-Selectin analysis (Example 1) . As a starting point for P-Selectin mutagenesis we generated a three dimensional model of the P-Selectin lectin domain in the same manner that the E-Selectin model was generated. Comparison of the two models revealed that of the residues that appeared most important for E-Selectin binding- to 2'3 sLe x , three are conεerved in P-Selectin: Y48, Kill and K113. In E-Selectin, the εubεtitutions Y48F, KlllA and K113A each profoundly decreased εLe x binding. Mutation of position 84 from R to A did not affect sLe x binding by E-Selectin, and mutation of position 8 from E to A increased sLe x binding by E-Selectin. Figure 14 shows the effect of complimentary εubεtitutionε at these poεitionε in P-Selectin on the binding of the anti-P-Selectin mAb's. Whereas none of these εubstitutionε significantly affected capture by the nonblocking antibodies (AC 1.2 and CRC 81), each of the subεtitutions K8A, KlllA and K113A partially decreased binding of the blocking antibody AK-6 (Figure 14) . These results are consistent with the PE-l chimera resultε above which localized part of the AK-6 epitope to the lectin domain of P- Selectin. Theεe results are also consistent with the relatively close alignment of these three positionε along the same face of the P-Selectin lectin domain. Furthermore, the complimentary εubstitutionε K8A and K113A in E-Selectin completely aboliεhed binding of a number of blocking mAb'ε to E-Selectin. Alεo like E-Selectin, mutation of the residues at positions 48 and 84 in P-Selectin did not affect mAb binding (Figure 14) . Next, we evaluated these P-Selectin mutantε for binding to immobilized glycolipidε and cells (Figure 15) . Measurement of the binding of this panel of mutants to the 2'3 sLe* glycolipid indicated that P-Selectin appears to use some of the same residueε aε E-Selectin in binding this carbohydrate (Figure 15A) . Whereas P-Selectin mutants with the subεtitutionε K8A and K84A εtill bound 2'3 sLe ' , the mutantε Y48F and K113A were completely negative. In E-Selectin the mutant KlllA did not bind 2'3 εLe x at all. Here, however, the P-Selectin mutant KlllA mediated partial binding to 2'3 εLe " , perhaps indicating a subtle difference in recognition of this εugar by E- and P-Selectin. A different εet of residueε appeared to be important for binding to the 2'6 form of sLe x (Figure 15B) . The subεtitutions K8A, KlllA and K113A ablated binding, while Y48F had no effect. The mutant K84A alεo εtill bound 2'6 εLe 1 (Figure 15B) . When εulfatide binding waε evaluated, a third pattern emerged (Figure 15C) . Only the mutation K113A significantly decrease εulfatide binding by P-Selectin. Theεe reεultε indicate that the same face of P-Selectin appears to participate in binding these thre glycolipidε, with εubtle differenceε in the reεidueε uεed to bind eac εugar. Since a more relevant aεεay for measuring P-Selectin interaction with its ligand is the cell binding assay, the panel of mutants wa evaluated by flow cytometry for εtaining of HL60 cells (Figure 14D) . Interestingly, the binding pattern seen with cells closely mimics that εeen with the immobilized glycolipid 2'3 εLe 1 - K8A and K84A both bound to HL60 cells, Y48F and K113A did not, and KlllA bound HL60 cellε only partially. Similar reactivities were εeen when neutrophils were stained (data not shown) . So, mutation of reεidues within this pocket of P- Selectin alεo affected binding to itε cognate ligand on cells- Furthermore, comparison of the reactivity of thiε panel of mutants with purified glycolipids provided some potential insights into the nature of the carbohydrate εeen by P-Selectin (see Discussion) . In E-Selectin the arginine at position 97 waε alεo important for εugar recognition. Mutation of thiε reεidue to alanine completely aboliεhed E-Selectin/2'3 εLe x binding (Example 1) . The residue at position 97 in P-Selectin is a serine and the above resultε indicated that P-Selectin appears to use the same region as E-Selectin in binding to itε ligand. Therefore, we teεted if this difference in residueε at poεition 97 could account for the differences in ligand binding by E- and P-Selectin. Examination of the three dimensional models of the E- and P-Selectin lectin domains reveals that amino- acid 97 falls within a loop formed by residues 94-100, which iε an inεertion in εelectinε relative to the mannoεe binding protein- The εequence of these two selectins is quite different through thiε stretch- YIKREKDV for E-Selectin vε. YIKSPSAP for P-Selectin- εo these loops would be expected to have different con ormations. To test the importance of the residue at poεition 97 in conferring εpecificity to εelectins, we made a P-Selectin- IgG mutant with the 94-100 loop replaced with the corresponding residueε from E-Selectin: S97R, P98E, S99K, A100D, P101V. We then teεted this mutant (abbreviated REKDV) for binding to antibodieε, glycolipidε and cellε. Binding of the P-Selectin-IgG REKDV mutant to each of the three anti-P-Selectin mAb'ε (AK-6, AC 1.2, and CRC 81) was approximately 70% of control P-Selectin-IgG binding. This would seem to indicate that although folding of thiε mutant is largely correct, some subtle structural perturbations may be present. Accordingly, this mutant did not bind any of the purified glycolipids (data not shown) . However, the REKDV mutant did bind to HL60 cells, although its binding waε significantly lesε than that εeen with control P-Selectin-IgG (70% cells positive, MFI 290 for REKDV mutant vs. 97% cellε poεitive, MFI 416 for control P-Selectin-IgG) . Thuε, tranεferring thiε loop (containing residue 97) from E-Selectin to P-Selectin did not completely disrupt the ability of the resultant P-selectin mutant to recognize its cellular ligand. These resultε would seem to imply that at least some of the differences in binding between E- and P-Selectin muεt be due to differenceε outside of this region (see Discuεεion) . Discussion Reεearch on selectin-carbohydrate interactions continues to be hampered by a lack of detailed underεtanding of the sugar structures seen by each -adhesion molecule. However, resultε from a number of approaches, including direct binding εtudieε, εoluble carbohydrate inhibition studies, and structural and conformational analyseε of purified potential ligands, have indicated commonalities in selectin recognition. Many of theεe findingε have centered around the sLe x core εtructure. However, many of these proposed similarities may be artifacts of forced binding under experimentally manipulated circumstanceε (εee Varki, A., Cur. Qpin. Cell Biol. 4.: 257-266 (1992) for discussion) . In vitro assays with solid phase carbohydrate ligands and transfected, over expresεed selectins can be misleading due to the unnaturally high densities of both receptors and ligands (Varki, A., Supra) . Furthermore, unrelated sugars can inhibit the same lectin interaction due to structural mimicry (Varki, A., Supra) . The flow cytometric assay employed here to measure P- Selectin'£3 interactions with its cellular ligand should avoid most of these limitationε while still being senεitive and convenient. The experimentε preεented here indicate that the measured binding observed using the P-Selectin-IgG chimera to εtain cellε accurately repreεentε thiε interaction. Studies to date have shown that P-Selectin binds a single, possibly unique, major glycoprotein of 120 Kd (Cummingε, R.D. et al. , ■ Cell Biol. 118: 445-456 (1992)) . The εame glycoprotein haε been iεolated from both neutrophilε and HL60 cellε (Cummings, R.D. et al . , Supra) and the number of εuch binding εiteε for P-Selectin iε eεtimated at 10,000-20,000 per cell (McEver, R.P. et al., J. Cell Biol. 112: 491- 499 (1991), Cummings, R.D. et al . , Supra, Berndt, M.C., J. Biol. Chem. 266: 5371-5374 (1991)). sLe " probably forms some component of this glycoprotein ligand, and sLe x iε εufficient to confer εome P-Selecti binding. However, sLe ' iε not sufficient to confer the saturable, hig affinity binding characteristic of P-Selectin adheεion (McEver, R.P. e al . , Supra) . Therefore, the P-Selectin ligand muεt have εtructura featureε in addition to sLe x that confer specificity and affinit (Cummings, R.D. et al . , Supra; R.D. Cummings, J. Cell. Biol. 115: 557- 564 (1991)) . The protein portion of the P-Selectin ligand may contribute to thi specificity and affinity by: (a) presenting the sugar in the correc configuration, (b) presenting multivalent sugarε to enhance bindin avidity, ' and (c) participating in a protein/protein contact with P Selectin (Cummingε, R.D. et al., Supra) . In fact, a role fo preεentation of polyvalent ligands to L-Selectin by the GlyCAM 1 ligan has already been proposed (Dowbenko, D. et al., Cell 69: 927-93 (1992) ) . In the aεεay described here, P-Selectin-IgG binding was ablate by protease treatment of cells, consiεtent with a requirement for thi glycoprotein (Ahern, T.J. et al., J. Biol. Chem. 267: 11104-1111 (1992)) . Aε noted above, sialic acid is crucial to P-Selectin bindin and εialidase treatment also abolished binding- Most importantly, removal of calcium chelation by EGTA also led to a losε of binding, a result that is a signature of the biologically relevant interactions performed by all C-type lectinε (Drickamer, K. et al.. Supra) . A suprising finding was that the E-Selectin-IgG chimera did not bind HL60 cells or neutrophils in this fluid phase staining asεay. Thiε iε deεpite the fact that the E-Selectin carbohydrate ligand, εLe x , iε clearly expreεsed by these cells (Gaeta, F.C.A., Science 250: 1130-1132 (1990); Aruffo, A., Science 250: 1132-1135 (1990)). Furthermore, we and others (Alford, J. et al. , J. Leuk. Biol. 52: 85-88 (1992)) have found that E- Selectin-IgG iε capable of binding HL60 cellε and neutrophils when the chimera is presented on a solid substrate, suggesting that the lack of binding in the fluid phase may be due to lower affinity of E-Selectin for itε cognate cell εurface ligand. Thuε, E-Selectin and P-Selectin are clearly distinct in binding to cells both as soluble Ig chimeras, as well as when they are expressed on endothelial/platelet cell surfaces. At least part of this difference between E- and P-Selectin must be due to differences in their lectin domains. Transferring the P-Selectin lectin domain onto the E-Selectin-IgG construct resulted in a molecule (PE-l) which stained cells, albeit at a lower intensity than P-Selectin- IgG. Carbohydrate reactivity was completely transferred with the relevant lectin domain- Thus, PE-l reacted with the purified glycolipids in a manner that was indistinguishable from P-Selectin-IgG and quite distinct from E-Selectin-IgG. Therefore, the lectin domain of each selectin appears sufficient for determining the differences in reactivities with theεe relatively small sugars. This result is consiεtent with a study by Kansas et al. (Kansaε, G. S., et al. , J. Cell Biol. 114: 351-358 (1991)) in which domainsof L- and P-Selectin were exchanged to show that PPME and fucoidin binding, both L-Selectin- εpecific carbohydrate ligandε, aε well aε the epitope defined by blocking mAb LAM1-3, map at leaεt in part to the C-terminal 67 amino acid reεidues of the L-Selectin lectin domain. Theεe authors alεo demonstrated that the CR domainε are not important for conferring PPME or fucoidin εpecificity (Kansas, G.S. et al., Supra) . The EGF and CR domains of selectins have clearly been εhown to perform vital εtructural roleε for theεe receptorε (Bowen, B. e al., J. Cell. Biol. 107: 1853-1862 (1990); Jutila, M.A. et al., J. Exp. Med. 175: 1565-1573 (1992); Aruffo, A. et al., Science 250: 1132-1135 (1990); Fennie, C. et al., J. Cell. Biol. 115: 235-243 (1991) ) . Whether theεe domains in P-Selectin also participate in making crucial contacts with its glycoprotein ligand cannot be answered here. However, the results of this study do place limitations on the nature of any such contacts. First, the P-Selectin- IgG chimera employed here only contains the lectin, EGF and CR1 domains of P-Selectin (Dowbenko, S. et al.. Supra) . Thus, CR2-CR9 must not form necessary contacts for the high affinity binding between P-Selectin and its ligand and it is interesting to note that mouεe P-Selectin lacks the CR2 domain (Isenmann, S. e al., J. Biol. Chem. 267: 15176-15183 (1992) . In addition, because the PE-l chimera did bind cells, any potential protein/protein contact sites may map to the lectin domain of P-Selectin. The difference in staining between PE-l and P-Selectin-IgG might reflect subtle conformational effects of the P- or E-Selectin EGF domains interacting with the common lectin domain. However, it iε important to εtreεs that protein/protein contacts mediated by the EGF or CR1 domains cannot be ruled out. Two recent studies have identified regions of the P-Selectin lectin domain that may be important for cell adhesion. Geng and coworkers showed that a mAb capable of inhibiting neutrophil binding to P-Selectin mapped to residueε 19-34 of thiε molecule, and that a peptide correεponding to this stretch also inhibited neutrophil binding to P- Selectin (Geng, J.G., et al., J. Biol. Chem. 266: 22313-22318 (1991)). This group described other peptides from the lectin domain of P-Selectin (corresponding to residues 23-30, 54-63, and 70-79) which blocked P- Selectin-mediated adhesion (Geng, J.G., e al., J. Biol. Chem.. "Lectin- domain peptides from selectinε interact with both cell εurface ligandε and Ca2+ ions," in press (1991). In the model of P-Selectin, these residues fall on the opposite εide of the lectin domain from the εit that we have identified aε important for εelectin-carbohydrate bindin and cell adheεion (see Example 1) . The residueε characterized by Gen and co-workers may represent a second εite in P-Selectin which may bin the carbohydrate and/or protein component of itε ligand. In light o this, it is important to remember that the resultε with the REKDV mutan indicated that not all of the differenceε in εpecificity between E- an P-Selectin can be explained by the region identified in thiε study. Therefore, the possible co-operation of this site with those describe by Geng et al. in conferring P-Selectin binding specificity warrant exploration. The results presented here eεtabliεh that the site previousl identified as crucial for E-Selectin binding to 2'3 sLe x is alεo crucia to P-Selectin binding to this ligand. Mutations in two of the conserve residueε within this εite, Y48 and K113, completely abolished 2'3 sLe binding and cell adheεion by both E-and P-Selectin. The anti-P-Selecti blocking mAb AK-6 mapped to thiε εame εite, aε did all of the anti-E Selectin blocking mAb's. Furthermore, Mel-14, a mAB that blocks L Selectin-mediated adhesion in vitro and in vivo, maps to thiε regio (Bowen, .B. et al., Supra. The fact that adheεion blocking mABε to al three εelectinε bind to residues within this εite emphaεizes it importance to the adhesive functions of theεe proteins. By comparing the binding of the panel of P-Selectin mutants to 2' sLe', 2'6 εLe x and sulfatides with their ability to bind cells, som insight as to the nature of the carbohydrate component of the P-Selecti ligand can be gained- As noted above, one εtudy haε εhown that E- and P-Selectin have related but diεtinct carbohydrate εpecificitieε (Ahern, T.J., Supra) . For example, theεe authorε found that the interaction of E-Selectin with the sLe* component of the P-Selectin ligand precludeε P- Selectin binding (Ahern, T.J., Supra) . Uεing a 2'6 εialyl-εpecific lectin to block P-Selectin binding, they alεo propoεed that the P- Selectin ligand may contain a bidentate carbohydrate εtructure with one arm containing 2'3 sLe x and the other a terminal sialyl-2'6 beta Gal (Dell, A., et al., J- Biol. Chem- 259: 10925-10935 (1984); Ahern, T.J., Supra) . However, our results with the P-Selectin mutants would seem to question a role for 2'6 linked εialic acid in cell adhesion. The mutant K8A did not bind the 2'6 form of sLe x at all, but still bound the P- Selectin ligand on cellε- Furthermore, the Y48F mutant did not bind cellε at all, but εtill bound 2'6 sLe x . Consequently, 2'6 sLe x binding did not correlate with ligand binding. However, the binding to 2'6 εLe x in the εolid phase asεays employed here is weak compared to 2'3 sLe x and sulfatide binding, so caution iε warranted in interpreting theεe results. Thus, it is conceivable that presentation of the 2'6 sialylated carbohydrate to P-Selectin provideε a critical parameter of ligand recognition that iε not replicated in our εolid phase assay (Dowbenko, D. et al., Supra) . A εecond binding activity of P-Selectin whose biological relavance has been recently questioned is itε interaction with εulfatides. Sulfatide binding by P-Selectin iε probably not relevant in vivo due to the observation that thiε interaction is not calcium dependent, that it is not removed by proteaseε, and that cellε expreεsing sulfatideε (erythrocyteε and plateletε) do not necessarily bind P-Selectin (R.D. Cummings, et al., J. Cell. Biol. 118: 445-456 (1992)) . Alεo, εulfatide binding by the P-Selectin mutants studied here did not correlate with cell binding. For example, mutant Y48F bound sulfatides well but did not adhere to cells at all. Cell binding waε only correllated with binding to 2'3 εLe x . Each mutant which bound 2'3 εLe x bound cellε (K8A and K84A) , while thoεe which did not bind 2'3 sLe x (Y48F and K113A) did not bind cells, and one mutant (KlllA) showed partial binding to both 2'3 sLe x and cellε. This is interesting given a recent study demonstrating that expression of 2'3 sLe x correlated with a cell'ε ability to bind activated plateletε via P-Selectin (De bruijne-Admiraal, et al.. Supra) , and it iε conεiεtent with the mAB and carbohydrate blocking εtudies of Polley et al. (Hakomori, S. et al., Proc. Natl. Acad. Sci. USA 88: 6224-6228 (1991)). Although one cannot rule out the involvement of 2'6 linked sialic acid or sulfatides in P-Selectin's interactions with its ligand, the data presented here clearly questionε the role (if any) they may play. Specificity of binding between E- and P-Selectin may derive from the manner in which 2'3 sLe x is presented (ie. glycolipid vs glycoprotein) . However; it must be allowed that 2'3 εLe " may not be the naturally occuring carbohydrate ligand recognized by either selectin, and that these differences in εelectin binding could be accounted for by subtle changes in the saccharide itεelf (Varki, A., Supra) . Sulfatides, as well aε the sulfated glycans heparin, fucoidin, and dextran sulfate, may inhibit P-Selectin function by mimicking its ligand (R.D. Cummings, et al . , J. Cell Biol. 118: 445-456 (1992)) . sLe\ sulfated glycans, and sulfatideε all have a negative charge which may play a role in the interaction of P-Selectin with itε ligand (De bruijne-Admiraal et al . , Supra; Example l) and theεe εugarε may inhibit εelectin-mediated adheεion by binding to a common εite (for example, at K113) which iε important for P-Selectin/ligand interaction . Example 4 cDNA εequences encoding the entire extracellular portion of human E-selectin (amino acids IM to 532S) were fused to the carboxy terminal 37 amino acids of CD16 hich contains the εignal εequence for cell surface anchorage via a glycosylphosphatidylinositol linkage (GPI) . The fuεion construct was cloned into the plasmid vector pEF-Boε [Mizushima and Nagata, Nucl. Acids Reε. 18, 5322 (1990)], and the GPI anchored E- εelectin expreεεed under the control of the human EF-lalpha chromosomal promoter. Point mutations were introduced into this cDNA construct using standard oligonucleotide directed mutagenesiε techniques, as described in Example 1. Transient expresεion in COS-7 cellε waε uεed to addreεε the impact of each E-εelectin mutation on structure/function. The function of mutants E-εelectinε waε determined by performing adheεion aεsays with human neutrophilε aε described in Example 3. The following mutations were found to aboliεh neutrophil binding without altering their recognition by the panel of Mabε: Y48F, Y94F, R97A, Kllla, and K113A E-εelectin. A mutation at poεition 105 (N105D) εhowed a marked reduction in binding of monoclonal antibodieε specific for the lectin egf domain, suggeεting a εever εtructural perturbation. In addition, E8A, and R84,K86A exhibited an increaεed neutrophil binding capacity. Theεe results are in agreement with the results obtained by the sLex binding aεsay disclosed in Example 1. Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments without diverting from the overall concept of the invention. All such modifications are intended to be within the scope of the present invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Genentech, Inc., F.Hoffmann-La Roche AG (ii) TITLE OF INVENTION: Selectin Variants (iii) NUMBER OF SEQUENCES: 35 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Genentech, Inc. (B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: USA (F) ZIP: 94080 (v) COMPUTER READABLE FORM. (A) MEDIUM TYPE: 5.25 inch, 360 Kb floppy diεk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: patin (Genentech) (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C). CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 07/879036 (B) FILING DATE; 30-APR-1992 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Dreger, Ginger R. (B) REGISTRATION NUMBER: 33,055 (C) REFERENCE/DOCKET NUMBER: 761P1 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415/266-3216 (B ' ) TELEFAX: 415/952-9881 (C) TELEX: 910/371-7168 (2) INFORMATION FOR SEQ ID NO.l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATAAGTCATA GCGGCCGTGG AGGTGTT 27 (2) INFORMATION FOR SEQ ID N0:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CTCATTATAA GTGGCAGCTT CCGTGGA 27 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baεeε (B) TYPE: nucleic acid (C) STRANDEDNESS: εingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ACTGGCCTCA TTGGCAGTCA TAGCTTC 27 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baseε (B) TYPE: nucleic acid (C) STRANDEDNESS: εingle (D)- TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATAAGCACTG GCTGCATTAT AAGTCAT 27 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baεeε (B) TYPE: nucleic acid (C) STRANDEDNESS: εingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTCAATCTCT TCTGCGTTTT GAATTGC 27 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACTTGGTGAA TAGGCCAATA TGGAGTT 27 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baseε (B) TYPE: nucleic acid (C) STRANDEDNESS: εingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATAACTTGGT GAAGCGCTCA ATATGGA 27 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baεeε (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GTAATAACTT GGTGCATAGC TCAATAT 27 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baseε (B) TYPE: nucleic acid (C) STRANDEDNESS: εingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CCAGTAATAA CTTGCTGAAT AGCTCAA 27 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baεes (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: AATCCAGTAA TAAGCTGGTG AATAGCT 27 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TCCAATCCAG TAAGCACTTG GTGAATA 27 (2 ) INFORMATION FOR SEQ ID NO : 12 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GATTCCAATC CAGGCATAAC TTGGTGA 27 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 baεeε (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TTCTTCTGTC AGAGGCGCCT GGGTTCCTAC CCA 33 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGGTTCACCT GGCGCCCAGT TCGCGGCTTC TTCTGT 36 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CACGCAGTCC TCATCTGCTT GCGCATTGTT GGGTTCACC 39 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baseε (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO-.16: CTTGATGTAG ATGGCCACGC AGTCCTC 27 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TCTCTTGATG TAGGCCTCCA CGCAGTC 27 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TTCTCTCTTG ATGGCGATCT CCACGCA 27 (2) INFORMATION FOR SEQ ID NO 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY, linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TTTTTCTCTC TTGGCGTAGA TCTCCAC 27 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: ATCTTTTTCT CTCGCGATGT AGATCTC 27 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baseε (B) TYPE: nucleic acid (C) STRANDEDNESS: εingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CACATCTTTT TCAGCCTTGA TGTAGAT 27 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GCCCACATCT TTGGCTCTCT TGATGTA 27 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baseε (B) TYPE: nucleic acid (C) STRANDEDNESS: εingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23 CATGCCCACA TCGGCTTCTC TCTTGAT 27 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baseε (B) TYPE: nucleic acid (C) STRANDEDNESS: εingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24. CCACATGCCC ACGGCTTTTT CTCTCTT 27 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baεeε (B) TYPE: nucleic acid (C) STRANDEDNESS: εingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 25 : ATTCCACATG CCAGCATCTT TTTCTCT 27 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baεeε (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CTCATCATTC CATGCGCCCA CATCTTT 27 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CTTGCTGCAC CTCGCATCAT TCCACAT 27 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GGCAAGCTTC TTGGCGCTGC ACCTCTC 27 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GCATAGGGCA AGAGCCTTCT TGCTGCA 27 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 baseε (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: AGCTTCCGTG GAGTAGTGGT AAGTCCAGGC TCCACT 36 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: ATTATAAGTC ATATTTTCCG CGGAGGTGTT GTA 33 (2) INFORMATION FOR SEQ ID NO:32; (i) SEQUENCE CHARACTERISTICS: (A. LENGTH: 33 bases (B TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: ACTGGCCTCA TTCCAACTGT AAGCTTCCGT GGA 33 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: ATTCCACATG CCCGTATCTT TTGGTCTCTT GATGTA 36 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CCAGTTCATG GGGGCTTCAG AATAATG 27 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: CCATGAGTAT GCAGCTGTGC TGTAATG 27

Claims: CLAIMS: 1. A selectin variant having an amino acid alteration at a site or sites within a patch defined by amino acid residue numbers 1-9, and 90 to the C-terminus in the three dimensional structure of the lectin domain of the corresponding native selectin. 2. The variant of claim 1 wherein the alteration iε within the three dimenεional patch defined by amino acid residue numbers 7-9, 90- 109, and 113 of the lectin domain of the corresponding native human selectin. 3. The variant of claim 1 wherein the alteration is within the three dimensional patch defined by amino acid residue numbers 7-9, 43- 48, 82-86, 94-100, 111 and 113 of the lectin domain of the corresponding native human selectin. 4. The variant of claim 3 wherein the alteration iε at one or more of amino acid poεitionε 7-9, 43-48, 82-86, 94-100, 111 and 113 of the correεponding native human εelectin. 5. The variant of claim 4 wherein the alteration is at one or more of amino acid positions 7-9, 47, 48, 82, 84, 86, 94, 97, 99, ill and 113 of the corresponding native human selectin. 6. The variant of claim 5 wherein the alteration is at amino acid residue numbers 7-9, or 84-86 of the corresponding native human selectin. 7. The variant of claim 6 wherein the alteration is a εubεtitution. 8. The variant of claim 7 that iε an E- or L-Selectin variant and has an uncharged amino acid εubεtituted for a charged amino acid at amino acid reεidue number 8 of the lectin domain of the correεponding native human selectin. 9. The variant of claim 8 wherein the amino acid εubstituted iε alanine, valine, serine or threonine. 10. The variant of claim 9 wherein the amino acid substituted is alanine. 11. The variant of claim 10 that is an E-Selectin variant and has alanine substituted for glutamic acid at amino acid position 8 of the native human E-Selectin lectin domain. 12. The variant of claim 10 that is an L-Selectin variant and has alanine εubεtituted for lyεine at amino acid poεition 8 of the native human L-Selectin lectin domain. 13. The variant of claim 7 that iε an E- or L-Selectin variant and has an uncharged amino acid εubεtituted for a charged amino acid at amino acid residue numbers 84 and 86 of the lectin domain of the corresponding native human εelectin. 14. The variant of claim 13 wherein the amino acid εubεtituted is alanine, valine, serine or threonine. 15. The variant of claim 16 wherein the amino acid substituted is alanine - 16. The variant of claim 15 that is an E-Selectin variant and has alanine substituted for arginine at amino acid position 84, and alanine substituted for lysine at amino acid position 86 of the native human E-Selectin lectin domain- 17. The variant of claim 7 having a positively charged amino acid at at least one of the amino acid poεitionε 111 and 113 of the lectin domain of the correεponding native human selectin. 18. The variant of claim 17 having a positively charged amino acid at both of the amino acid positionε 111 and 113. 19. The variant of claim 18 wherein εaid positively charged amino acid is lysine or arginine. 20. The variant of claim 19 having retained the native amino acids at positions 111 and 113 of the lectin domain of the corresponding native human εelectin. 21. -The variant of claim 20 that iε an E- or L-Selectin variant and has a positively charged amino acid at each of the amino acid poεitionε 97, ill and 113 of the lectin domain of the correεponding native human selectin. 22. The variant of claim 21 having retained the native amino acidε at positions 97, 111 and 113 of the corresponding native human εelectin. 23. The variant of claim 6 having retained the epidermal growth factor (egf) like domain and the complement binding domains of the corresponding native human selectin- 24: The variant of claim 23 that is E8A E-Selectin, K8A L- Selec in, or R84A,K86A E-Selectin. 25. A DNA sequence encoding any of the variants of claims 1, 6, 8, 13 and 24. 26. A replicable expression vector containing and capable, in a transformant host cell, of expressing the DNA sequence of claim 31. 27. A host cell transformed with the vector of claim 26. 28: The host cell of claim 27 that is eukaryotic. 29. The hoεt cell of claim 28 that iε mammalian. 30. A method compriεing: (a) introducing an amino acid alteration into the lectin domain of a selectin; and (b) εcreening the reεultant εelectin variant for increaεed affinity to a native εelectin ligand. 31: The method of claim 30 wherein εaid alteration is introduced within the three dimensional patch defined by amino acid residue numbers 7-9, 90-109, and 113 of the lectin domain of the corresponding native human selectin. 32- A pharmaceutical composition useful for the treatment or prevention of a εymptom or condition aεεociated with a pathologic inflammatory reεponse mediated by a selectin, comprising a therapeutically or prophylactically effective amount of the εelectin variant of claim 1 in admixture with a pharmaceutically acceptable carrier. 33. A method for the treatment or prevention of a symptom or condition asεσciated with a pathologic inflammatory reεponεe mediated by a εelectin compriεing administering to a patient having or at risk of developing such symptom or condition an effective amount of the composition of claim 32. 34. A chimeric protein compriεing a fusion of an amino acid sequence comprising a selectin lectin domain sequence having an amino acid alteration at one or more sites within a patch defined by amino acid residue numbers 1-9, and 90 to the C-terminuε of the lectin domain of the corresponding native εelectin to an immunoglobulin constant domain sequence. 35. The chimeric protein of claim 34 comprising selectin egf like domain and complement binding domains. 36. An anti-E-Selectin monoclonal antibody composition capable of binding to εubstantially the same E-Selectin lectin domain epitope recognized by, or which compete with, a monoclonal antibody selected from the group consisting of 7H5; 8E4; 3B7; 1D6; 4D9; 1E5; 9A1; 7E10; 1B3; 14G2; 11G5; and 9H9. 37. The monoclonal antibody composition of claim 36 capable of binding to subεtantially the same E-Selectin lectin domain epitope recognized by, or which compete with, a monoclonal antibody selected from the group consisting of 7H5, 8E4 and 3B7. 38. The monoclonal antibody composition of claim 37 wherein said epitope includes the amino acids at poεitionε 7 and 9 of human E- εelectin. 39. The monoclonal antibody compoεition of claim 38 wherein said epitope ' further includes amino acid position 113 of human E-selectin. 40. The monoclonal antibody compoεition of claim 39 wherein εaid epitope further inlcudeε amino acid poεitions 98 and 101 of human E- selectin.

Inventors: BURNS DANIEL K, ERBE DAVID V, LASKY LAURENCE A, PRESTA LEONARD G, WOLITZKY BARRY A, BURNS, DANIEL, K., ERBE, DAVID, V., LASKY, LAURENCE, A., PRESTA, LEONARD, G., WOLITZKY, BARRY, A.
Applicants: GENENTECH INC, HOFFMANN LA ROCHE, F.HOFFMANN-LA ROCHE AG, GENENTECH, INC.
Application year: 1993
Main classification: C12N15
IPC codes: A61K38, A61P17, A61P19, A61P25, A61P29, C07K14, C07K16, C07K19, C12N1, C12N5, C12N15, C12P21, C12R1