CA2224625A1

CA2224625A1 - P-selectin ligands and related molecules and methods

Info

Publication number: CA2224625A1
Application number: CA002224625A
Authority: CA
Inventors: Brian Seed; Tara Pouyani
Original assignee: Individual
Current assignee: General Hospital Corp
Priority date: 1995-06-14
Filing date: 1996-06-11
Publication date: 1997-01-03
Also published as: JPH11508131A; EP0833650A4; BR9608918A; IL122590A0; AR003436A1; HUP9901131A2; WO1997000079A1; NO975862D0; TR199701620T1; KR19990022954A; NO975862L; AU6272996A; CZ401497A3; EP0833650A1; ZA965032B

Abstract

Disclosed herein are organic molecules to which are covalently bonded a sialylLex determinant and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule. Also disclosed are particular P-selectin ligands and P-selectin ligand-antibody fusions. These moecules, ligands, and fusion proteins find use in methods of reducing or protecting against inflammation and extravasation-dependent adverse reactions, such as organ damage and clotting (for example, associated with adult respiratory distress syndrome or ischemic myocardial injury).

Description

W O 97/00079 PCTrUS96/10043 P--S~T-~CTIN LIGA~nDS
A~nD R~T.~n MOLEClnLES l~ND ~EI~HODS
St~tement as to Federally S~onsored Research sThis invention wa3 made with Government ~o~L
under NIH grant DK43031, and the Government therefore has certain rights in this invention.
Bac~Lou~ld of the Invention This invention relates to P-selectin ligand 10 molecules, DNAs, and uses thereof.
P-selectin i8 an integral membrane C--type lectin found within the Weibel-Palade bodies of endo~h~~
cells and the alpha granules of platelets (McEver et al., J. Clin. Invest., 84:92-99, 1989; Bonfanti et al., 15 Blood, 73:1109-1112, 1989; Hsu-Lin et al., J. Biol.
Chem., 259:9121-9126, 1984; St~h~rg et al., J. Cell Biol., 101:880-886, 1985). Its translocation to the plasma membrane can be in~llce~ by thrombin, histamine and other -~Ators released by mast cell activation, 20 complement C5b-9 complex or C5a fragment, ~e~uxides, and QY;~ed low-density lipoprotein (Hsu-Lin et al-, J.
Biol. Chem., 259:9121-9126, 1984; St~nhe~g et al., J.
Cell Biol., 101:880-886, 1985; Hattori et al., J. Biol.
Chem., 264:9053-9060, 1989; Kubes and Kanwar, J.
25 Immunol., 152:3570-2577, 1994; Thorlacius et al., Biochem. Biophys. Res. Communications, 203:1043-1049, 1994; Foreman et al., J. Clin. Invest., 94:1147-1155, 1994; Patel et al., J. Cell Biol., 112:749-759, 1991;
Lehr et al., Laboratory Invest., 71:380-386, 1994;
30 Gebuhrer et al., Biochem. J., 306:293-298, 1995). Once displayed on the cell surface, P-selectin supports the attachment of myelomonocytes to platelets or endothelial cells (Larsen et al., Cell, 59:305-312, 1989; ~A hll~ger and Mc~ver, Blood, 75:550-554 1990; Geng et al., Nature, 35 343:757-760, 1990; Gamble et al., Science, 249:414-417, CA 0222462~ 1997-12-12 W O 97/00079 PCTrUS96/10043 1990). In the latter setting, its appearance heralds an underlying ~ insult and supports the initial ~tep ~n leukocyte extravasation, the rolling of neuLro~hils along the postcapillary venule wall (Lawrence and Springer, 5 Cell, 65:859-873, 1991). Mice which are homozygously deficient for the P-selectin structural gene exhibit decreased leukocyte rolling and show delayed recruitment of granulocytes to sites of experimentally ;n~llce~
inflammation (Mayadas et al., CQ11~ 74:541-554, 1993).
10 Generally, the mediators which ;n~llc~ P-selectin expression are involved in 8ign;-1ing trauma or wollnrlin~
One of the first reço~ni~e~ res~n~~- to ~ trauma is mast cell activation, which is accompanied by release of histamine, serotonin, and other diffusible mediators.
15 Other ~_ -~ events include thrombus formation at sites of vA~ A~ lu~Lu e and complement alternative pathway engagement by foreign bodies. P-selectin ~x~e~sion is ~n~ll~e~ by signals generated in each of these contexts.
Al~hs~yh induction of P-selectin mediated neu~.o~hil 20 rolling has been thought to be an inevitable con~ yuence of surgical intervention, cromolyn, an agent which blocks mast cell degranulation, has been shown to prevent such rolling, thereby providing an elegant ~r nctration of the role of the mast cell as the link between trauma and 25 extravasation (Kubes and Kanwar, J. Immunol., 152:3570-3577, 1994).
Sut~ary of the Invention In a first aspect, the invention features an organic molecule to which there is covalently hQn~e~ a 30 sialyl-LeX dete- ;nAnt and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule.
In a ~ecQ~A aspect, the invention features a P-selectin ligand selected from the group consisting CA 0222462~ 1997-12-12 W O 97/00079 PCTrUS96/10043 ~ ~?~tially of: (a) amino acids 21-57 of Fig. 8A and (b) amino acids 38-57 of Fig. 8A.
In a third aspect, the invention features fusion proteins that include a P-selectin ligand joined to an 5 antibody ~t ~in (for example, one or more of the hinge, CH2, ~nd CH3 domains).
In related aspects, the invention features purified nucleic acid ~co~;n~ ~ protein cont~n;ng sites for the attachment of a sialyl-LeX determinan~ and a 10 sulfated determinant, at least one of these determinants being positioned at a non-naturally o~ul.ing site on the protein; purified nucleic acid ~nco~ing any one of the P-selectin ligands of the invention; purified nucleic acid ~nco~ng a P-selectin-antibody ~usion protein; and 15 vector and recombinant cells including any of these nucleic acids. Also included in the invention is the use of P-selectin ligands or organic molecules bearing such ligands (if desired, in combination with other proteins such as antibody or ~l-acid gly~u~Lein domains) in the 20 manufacture of a medicament for the treatment of any of the conditions described below.
In another related aspect, the invention features a method of inhibiting the b~ n~ i ng of a cell bearing a P-selectin protein to a molecule or cell bearing a sialyl-25 LeX determinant and a sulfated determinant. The methodinvolves contacting the P-selectin protein-bearing cell with either an organic molecule bearing sialyl-LeX and ~ulfated determinants, at least one of these determinants being positioned at a non-naturally occurring site on the 30 molecule; a P-selectin-antibody fusion protein; or any of the P-selectin ligands of the invention.
In another related aspect, the invention features a method of re~llc; ng inflammation in a mammal involving administering to the patient a therapeutically-effective 35 amount of either an organic molecule bearing sialyl-LeX

CA 0222462~ 1997-12-12 W O 97/00079 PCT~US96/10043 and sulfated determinants, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any one of the P-selectin ligands of 5 the invention.
In yet another related aspect, the invention features a method of reducing or protecting a mammal against any extravasation-~r~p~n~nt adverse reaction (including, without limitation, extravasation-r~en~nt 10 organ damage and/or clotting associated with adult respiratory distress ~y~.d~me, glomerular nephritis, and ischemic myocardial injury). The ~ethod involves administering to the mammal a therapeutically-effective amount of either an organic molecule to which there is 15 covalently hQn~ a sialyl-LeX and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any ~f the P-selectin ligands of the invention.
In a final aspect, the invention features a met~od of reducing or protecting a mammal against an adver~e immune reaction, involving administQring to the mammal therapeutically-effective amount of either an organic molecule to which there is covalently hQ~ a sialyl-LeX
25 and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any of the P-selectin ligands of the invention. Preferably, this method involves treating the - -1 for an adverse immune reaction ~hich is in~l~ce~ by a microbial factor. such microbial factors include, without limitation, gram-negative bacteria lipopolysaccharides (LPS), peptidoglycans from gram-positive orgAni~ , -nnA~ from fungal cell walls, 35 polysaccharides, extracellular e~:ymes (e.g., ~L~e~,~e~inAre) and ~Qytn~: (e.g., toxic shock entero~Qyinc of staphyloro~ci). In other preferred emho~;ments~ the method involves treating a mammal for any adverse immune reaction which is i~Allce~ by a host factor. Such host 5 factor~ include, without limitation, metabolites of compl ~ent, lr; n;n, and coagulation systems, factors relea ed from stimulated cells (e.g., cytQkin~c such as interleukin 1 (IL-1) and tumor necrosis factor-~ (TNF)), enzym~ and Qy~ t~ from polymorrhon-~clear leukocytes (PMNs), vasopeptides (e.g., histamine), and products of the metabolism of ar~h;~o~;c acid. In other preferred embodiments, the adverse immune reaction is ;nAllce~ by recombinant TNF-~ or is in~ll~e~ by recombinant IL-l. In yet other preferred ~ ho~; ments, the adverse immune 15 reaction is septic shock or is septicemia.
In preferred emho~l; ments of each of the above aspects, the organic molecule or protein also inhibits the b~nAing of a cell bearing an E-selectin (ELAM-1) protein to a molecule or cell bearing a sialyl-LeX
20 dete- ;n~nt and thus inhibits E-selectin-mediated inflammation, extravasation-A~r~nAent adverse reactions, and adverse immune reactions; the sialyl-LeX and sulfated determinants are present on a P-selectin ligand consisting essentially of: amino acids 21-S7 of Fig. 8A
(for example, amino acids 38-57 of Fig. 8A); the sialyl-LeX determinant is N-linked or 0-l;nkeA; the molecule or protein contains multiple sialyl-LeX and/or multiple sulfated determinants; the organic molecule is a protein (for example, an antibody (for example, IgG or IgM), ~1-30 acid glycoprotein (AGP), or an antibody fusion protein(for example, an AGP-antibody fusion protein), the protein is an antibody, AGP, or an antibody fusion protein (for example, an AGP-antibody fusion protein) to which any of the P-selectin ligands described herein is 35 appended (for example, at the pro~in's amino-terminus);

, CA 0222462~ 1997-12-12 PCI/U~ 96 ~ Oa~3 IPEA~US 1 ~ J A N 1997 the antibody or antibody fusion protein (for example, the AGP-antibody ~usion protein) includes, as an antibody portion, an IgG1 CH2, CH3, and/or hinge domain; the antibody, AGP, or antibody fusion protein includes one or 5 more of the N-linked glycan addition sites of ~l-acid glycoprotein; the antibody portion o~ the molecule bears one or more non-naturally occurring sialyl-LeX
determinants; the sialyl-LeX determinant interferes with the antibody's ability to fix complement or bind an Fc 10 receptor (for example, due to a sialyl-~eX determinant attached to one or more of amino acids 274, 287, or 322 '~ of the sequence shown in Fig. lOA-E); and the organic ., "~ ..
molecule is soluble.
By a "P-selectin ligand", as used herein, is meant 15 any amlno acid sequence capable of mediating an interaction with the P-selectin receptor and includes those proteins referred to as P-selectin counter-receptors. Preferable P-selectin ligands include, without limitation, amino acids 21-57, and more 20 preferably amino acids 38-57, of Fig. 8A. P-selectin ligands according to the invention may be used in conjunction with additional protein domains (for example, antibody domains) to produce fusion proteins useful in the invention.
By ~non-naturally occurring" is meant a sialyl-LeX
or sulfated determinant that is not one which is naturàlly bound to the molecule at that amino acid location.
By ''inflammation" is meant a pathologic process 30 consisting of cytologic and histologic reactions that occur in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent.
Inflammation, as used herein, includes any acute inflammatory response (~or example, during or following AMEI\IDED S~lEET

W O 97/00079 PCTrUS96/10043 adult respiratory di~ ~Le~;S syndrome or ischemic myocardial injury) as well as any chronic inflammatory r~ron~e (for example, rheumatoid arthritis, psoriasi~, or pemphigus vulgaris).
S By "purified nucleic acid" is meant DNA that is free of the genes which, in the naturally-occurring genome of the organi~m from which the DNA of the invention is derived, flank the gene. The term therefore include~, for example, a recombinant DNA which i8 10 incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a pr4karyote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment prsAl~c~A by PCR or ~ LL iction enAon~lclease digestion) 15 ;nA~p~nA~nt of other seqU~nc~C. It also includes a recombinant DNA which is part of a hybrid gene ~n~oA i n~
additional polypeptide ~equence.
By "N-linked" is meant hQ~A~A to the amide nitrogen of an asparagine residue of a protein.
By ~O-link~" is mQant honA~A to the hydroxyl-group oxygen of a serine, threQn;n~, or hyd~ox~lysine re~idue of a protein.
By an "extravasation-A~r~nA~nt adverse reaction"
is meant any reaction which is detrimental to the host 25 and which results directly or indirectly from the ina~L OPL iate attachment of neutrophils to endothelium at or proximate to a site of inflammation, ti~ ~ damage, or thrombus formation and results in migration of those neuL.ophils into the at~A~h~ blood vessel or organ.
30 Organs which may be affected by such damage include, without limitation, the heart, lungs, and kidneys.
By an "adverse immune reaction" is meant any reaction mediated by an immune cell (i.e., any B cell, T
cell, monocyte/macrophage, natural killer cell, mast W O 97/00079 PCT~US96110343 cell, basophil, or granulocyte) and which is detrimental to the host.
ne~ Descr;~tion The drawings will first be briefly described.
Fig. lA is a schematic representation of the structure of the PSGL-1 deletion mutantD. Systematic deletion of the ectodomain of PSGL-1 was accomplished with ~v~l~ional PCR methods. A representative 10-residue repeat (stippled; SEQ ID N0:1) and the 10 transmembrane domain (hatched) are illustrated. Fig. lB
i8 histogram which represents P-selectin b; n~ i n~ activity of transfected COS cells expressing the deletions shown in Fig. lA. 51Cr-labeled cells were allowed to adhere to soluble P-Delectin adsorbed to microtiter wells. The 15 cells were washed, the bound cells were then lysed, and 51Cr levels were counted. Deletion constructs were illL.~ ce~ into cells either in the Ah~n~e (bar 2) or presen~e (remaining 7 bars) of the human FTVII
fucosyltransferase.
Fig. 2A is a schematic representation of chimeras of PSGL-1 and CD43. The membrane proximal extracellular domain, transmembrane, and intracellular domains of PSGL-1 were replaced with the cognate se~l~n~e~ of CD43.
The resulting molecule lacks cyst~n~c and thus cannot 25 form a ~ fide linke~ dimer. Fig. 2B is a histogram ~e~eDenting P-selectin b;n~;n~ activity of transfected CoS cells expressing the chimeras shown in Fig. 2A.
n, cotransfection with the human FTVII
fucosyltransferase.
Fig. 3A is a schematic representation of chimeric mucins bearing the PSGL-l apical domain appended to intact or truncated mucin C-termini. The PSGL-1 N-terminus (stippled; SEQ ID N0:1) and the tr~n~ ~mbrane (TM) domains (hatched) are illustrated. The sequence of 35 PSGL-l-NH2/CD43 "repeats" are represented by SEQ ID N0:2.

_ PSGL-l was fused to the N-terminus of the predicted mature CD34 and GlyCAM-1 molecules, and to the N-terminus of the repeat region of CD43. Fig. 3B is a hi~togram enting P-selectin bi nA i ng activity of transfected 5 CoS cells expressing the constructs shown in Fig. 3A.
h, human FTVII fucosyltransferase.
Fig. 4A is a schematic 1~ e~entation of PSGL
deletion mutants. The amino terminal domain was ~pre to PSG~ molecules having varying numbers of the repeated 10 element. Fig. 4B is a histogram r e~r ~~ ~nting P-selectin bi nA i n~ activity of transfected COS cells expressing the chimeras illustrated in Fig. 4A.
Fig. 5 is a photograph of an autoradiogram of mucin:immunogloh~ n fusion proteins labeled with 15 35S ~ fate and electrophoresed on an 8% denaturing polyacrylamide gel under reducing conditions. Lane A, supernatant of CDM8 transfected cells; Lane B, cupernatant of cells transfected with Ig expression vector (no mucin insert); Lane C, supernatant of cells 20 expressing PSGL-l:Ig; Lane D, supernatant of cells expres~ing CD43:Ig; Lane E, supernatant of cells expre~sing CD34:Ig; and Lane F, supernatant o~ cells expre~sing GlyCAM-l:Ig.
Fig. 6A and Fig. 6B are histograms representing 25 bi n~ ~ ng to immobilized P- and E-selectin of COS cells expressing PSGL-l with or without fucosyltransferase and in the ~-en~e or Ah~~n~e of 10 mM NaC103. Fig. 6A is a histogram representing binding of cells to P-selectin.
Fig. 6B is a ~to~am reprasenting bi n~; n~ of cells to 30 E-selectin.
Fig. 7 is a photograph of an autoradiogram of PSGL-l:immunoglobulin fusion proteins labeled with 35S-sulfate in the pr~Fen~e or Ah-~n~e of 10 mM NaC103 and electrophoresed on an 8% denaturing polyacrylamide gel 35 under reducing conditions. The photograph indicates that , CA 0222462~ 1997-12-12 P~TIUS 96 / 1 ~ a 4 3 IP~AIU~ 1 4 J A N ~997 chlorate inhibits incorporation of 35S-sulfate into soluble mucin chimeras. ~ane A, supernatant of CDM8 transfected cells in the absence of chlorate; Lane B, supernatant of cells expressing PSGL-l:Ig in the absence 5 of chlorate; Lane C, supernatant of CDM8 in the presence of chlorate; and Lane D, supernatant of cells expressing PSGL-l:Ig in the presence of chlorate.
Fig. 8A is a listing of the sequence endpoints of various PSGL-l deletion mutants (indicated by the arrows) 10 The uppermost sequence is SEQ ID NO:3; the middle sequence is SEQ ID NO:13; the lowermost sequence i8 SEQ
ID NO:14. Fig. 8B is a histogram representing P-selectin binding activity of transfected COS cells expressing the deletion mutants having the endpoints shown in Fig. 8A.
Fig. 9A is a schematic diagram of the constructs employed to measure the effect of appending wildtype and mutant variants of PSGL-1 residues 38-57 to deleted PSGL-1 or CD43. The inserted sequences are shown at bottom left. Fig. 9B is a histogram representing 20 P-selectin binding activity of transfected COS cells expressing the chimeras illustrated in Fig. 9A.
Fig. lOA-E are a listing of the nucleotide - sequence (SEQ ID NO:8) encoding IgG1 (SEQ ID NO:9) and mutations designed to create N-linked glycan addition 25 sites (SEQ ID NO:12).
Fig. llA-B illustrate the nucleotide sequence (SEQ
ID NO:10) and Fig. llC is the amino acid sequence (SEQ ID
NO:11) of an AGP-IgG1 fusion protein.
Fig. 12A is a schematic diagram of immunoglobulin fusion proteins consisting of either intact PSGL-1 tSEQ
ID NO:4) or 20 residue peptides joined to the hinge, CH2, and CH3 domains of human IgG1. Construct Y/F-hIgG bears SEQ ID NO 5; construct T/AhIgG bears SEQ ID NO:6;
construct Y/F-T/A-hIgG bears SEQ ID NO:7. Fig. 12B is a 35 photograph of an 8~ polyacrylamide gel used to assess A~ENDED SHEET

in~o-~ ation of [35S]cysteine and methionine by the fusion proteins shown in Fig. 12A following transfection into COS cell~. Lane A, supernatant of cells transfected with CDM8 control. Lane B, supernatant of cell~
5 transfected with PSGh-l-immunoglohlll in fusion protein.
Lane C, ~u~e~ ~-atant of cells transfected with WT-hIgG.
Lane D, supernatant of cells transfected with Y/F-hIgG.
~ane E, supernatant of cells transfected with T/A-hIgG.
Lane F, ~u~e~l~atant of cells transfected with Y/F-T/A-10 hIgG. Fig. 12C is a photograph of an 8% polyacrylamidegel used to AC-eC~ inc~ ation of [35S] sulfate by the fusion proteins shown in Fig. 12A following transfection of COS cells. In addition, a ~vllL~ol fusion protein bearing no amino-terminal addition was included (Lane B).
15 Lanes C through G correspond to Lanes B through F in Fig.
12B.
Fig. 13 is a bar graph of interacting HL-60 cells per video-ca~L~Led field. The cells were infused into a parallel plate flow chamber precoated with either P-~electin-immunoglQh~ n chimera or a CD4-immunglQhlll~n chimera col.L ~1. The cells were subjected to a shear stress of 0. 75 dynes/cm2. Each bar represents the average number of cells (tSEM) per field from eight frames taken at 15 ~~con~ intervals. Cells rolling or flowing appear a~ streaks on the video image. The bars represent, from left to right: HL-60 cells rolling or flowing over P-selectin-immunoglobin chimera, HL-60 cells pretreated in a sulfate-free medium with 10 mM sodium chlorate, and HL-60 cells flowing over CD4-immunoglobulin 30 chimera.

Sialyl-Lewis X (S3 alyl-LeX) and culfated determinants were shown to interact with P-selectin and facilitate bin~ing by the following experiments. These examples are presented to illustrate, not limit, the CA 0222462~ 1997-12-12 W O 97/00079 PCTrUS96/10043 invention. The methods used in the following experiments will first be described.
Pr~uc~-~on of Solllhle P-Select~n P-selectin and E-selectin Ig chimeras were 5 prepared by transient expression in COS cells of an expression plasmid encoAin~ the lectin, EGF-related, an~
first two short con~~ncus repeat related domains of P-selectin joined to the hinge, CH2, and CH3 domains of human IgGl (Aruffo et al., EMB0 J., 6:3313-3316, 1991;
10 Walz et al., Science, 250:1132-1135, 1990). The PSGL-l cDNA qo~i n~ sequence was ob~i n~ by PCR amplification of an HL-60 cDNA library, and the sequence confirmed by DN~
sequencing. The co~i ng segment for the mature extracellular, transmembrane, and intracellular domain 15 was inserted into an expression vector ba~ed on CDM8 which lacks the polyoma virus origin of replication and contains the leader sequence for the CD5 antigen positioned just ~-LLeam of the co~; n~ region for an influenza hemagglutinin (flu) peptide (Field et al., Mol.
20 Cell. Biol. 8:2159-2165, 1988) epitope tag.
Co~ tion of PSG-T-1 Delet;ons Amino terminal PSGL-1 deletion constructs were prepared by PCR amplification using primers en~oAi n~ tha desired endpoint of the deletion ~utant located 25 downstream of an XbaI site in frame two (~nco~ Leu Asp). The resulting se~l~nce~ enco~e~ a polypeptide in which the residues listed below i ~ tely followed the aspartic acid (D) of the Xba site: A118, A128, A138, A148, A158, A168, G178, A188, Al98, A208, A218, A228, 30 A238, A248, A258, and T268 of the PSGL-1 precursor. The PCR fragments were then inserted in the CD5 leader flu tag expression vector used for expression of the intact PSGL-l. The flu tag terminates in an XbaI site in the frame described above. Sequences at the flu tag junction 35 were verified, and expression was confirmed in CoS cells CA 02224625 l997-l2-l2 W 097/00079 PCTnUS96/10043 by indirQct immunofluore~c~n~ microscopy and flow cytometry. A series of internal deletions with an EcoRI
~ite at the ~ite of the daletion in frame one (oncoA~
glutamic acid phenylAlAnin~) was also prepared by first 5 creating deletion variants with amino termini (rQsidues immediately following phenylAlAninQ tF] of the EcoRI site aV~ ol~l;n~ to A118, A128, A138, A148, A158, A168, G178, A188, A198, A208, A218, A228, A238, A248, and A258 of the peptide sequence of the precursor. To each of 10 these deleted variants was appen~ a flu-tagged amino-terminal PSGL-1 domain en~in~ with an EcoRI site in the glutamic acid phenyl~l ~ni n~ frame immediately do~..~L~eam of PSGL--1 ~e~ ~or A117. The resulting constructs contained deletions between A117 and the various 15 Qndpoints above.
Muc; n Dc ~in Tnterchanges CD34, CD43, and GlyCAM-1 mucins were prepared for addition of the PSGL-l amino-terminal domain by Apre~ i n~
an EcoRI site to either the mature amino terminus (CD34 20 or GlyCAM-l), or to the beginning of a region of threonine/proline-rich repeats (CD43). As above, the EcoRI site was in the frame glutamic acid pheny~ n~ne (frame 1). The CD34 sequence began at residue F30 of the precursor, the Gly-CAM-l at precursor Ll9, and the CD43 25 at precursor I135. To each of these was app~n~ the flu-tagged PSG~-l domain terminating in EcoRI as above.
The amino terminus and repeat elements of PSGL-l were app~n~e~ to the membrane proximal, transmembrane, and intracellular domains of CD43 through an EcoRI site in 30 the glutamic acid phenylal ~n; ne frame positioned immediately ~eam of the se~nc~ S225 of the CD43 precursor. The complementary fragment from PSGL-l correspon~ to the amino-terminal residues of the precur~or up to T267.
35 Fine Structllre Ma~ing of ~he ~ino-Ter~in~l Dom~Ln W O 97/00079 PCT~US96/10043 ~ 14 ~
A similar strategy was employed for the construction of deletions in the amino-terminal domain, in which PCR generated deletions were formed using primers bearing an XbaI site in the leucine aspartic acid 5 frame (frame 2). Immediately downstream of the residues ~n~o~ ~ ng aspartic acid were the PSGL-1 se~l~ncefi ~oLLe~ ng to precursor R38, E58, P78, and A98. For the definition of the amino-terminal domain, duplex oligonucleotides were synthesized corre~pon~;n~ to the 10 residues between 38 and 57 with the indicated sequence changes to mutate threon;ne or tyrosine residues to ~lAn;~ or phenylAlAnlne. All constructs were confirmed by dideoxy sequencing.
Cell ~hesion AssaYs Transfected cells were de~Ach~ from culture h~C with 0.5 m~ EDTA in phosphate buffered ~ A1 in~
(PBS) 48 to 60 hours after transfection. The cells were then loaded with 100 ~1 51CrO4 (1 mCi/ml; DuPont, Boston, MA) in 0.9% NaCl plus 100 ml medium by inGllh~ting them ~t 20 37~C for 1 hour. TOAt1~1 cells were washed twice in PBS
and r~ cr~n~e~ in 0.2% BSA, 0.15 M NaCl, 3 mM CaC12.
Variation in labeling rate (counts incorporated per cell) between cells prepared in parallel with the same batch of labeled chromate was typically minimal. The labeled 25 cells were incllhAted in wells of 96-well microculture plates which had been coated with affinity purified goat anti-human IgG antibody (100 ~1 of 20 ~g/ml anti-human IgG Fc (heavy chain specific) in PBS) for 2 hours in a humid ch~mher at room temperature. After the plate was 30 washed twice with PBS, additional protein-b; n~; ng sitQs were blocked by an overnight ;n~lhAtion with 200 ~1 3%
BSA in PBS. The plate was washed with PBS four times and in~lhAted with 200 ~1 of fusion protein supernatants for 2 hours. Following three PBS washes and one additional 35 wash (in 0.2~ BSA, 0.15 M NaCl, 3 mM CaC12), 2 x 105 W O 97/00079 PCTrUS96/10043 cells/well (in 200 ~1 0.2% BSA, 0.15 M NaCl, 3 mM CaC12) were ~e~ and allowed to bind for lS minutes at room temperature while the plate rotated on a rotary platform (80 rpm). The plate was washed three times by f~ n~
5 the wells with 200 ~1 0.15 M NaCl/3 mM CaC12 and then inverting the plate. Adherent cells were lysed by the addition of 200 ~1 2% SDS, and labeled chromate was counted with a gamma ray spectrometer.
n~-f 1 llnrescence An;~ 1 VS i ~
Cells were prepared for cytometry by ;n~nh~tion with the primary monoclonal antibody (a 1:200 dilution of ascites or 5 ~g/ml of purified antibody is suitable) in PBS con~ining 3% BSA for 30 to 45 minutes. The cells were w~ twice with PBS and incllh~ted with 2 ~g/ml 15 FITC~conjugated affinity purified antibody tc eithQr mouse IgG (12CA5) or 0~ IgM (CSLEX-l) for 30 to 45 minuteC in PBS/3% BSA. ~he cells were then wA~s~ twice with PBS and resusr~nA~ in 1 ml of 1% freshly depolymerized paraformaldehyde in PBS prior to analysis.
20 For immunofluore~~en~e microscopy, transfected cells were fixed with 4% freshly depolymerized paraformaldehyde, WZ~ ~A~ ~!YpQ~~~ to BSA al: 3% in PBS for 30 minutes, and then incllh~ted with primary antibody (ascites, 1:250) for 30-45 minutes. The cellc were then washed twice with PBS
25 and ~ncl~h~ted for 30-45 minute3 with FITC-conjugated affinity-purified antibody to mouse IgG (Cappell; 2 ~g/ml in PBS cont~;n;n~ 3% BSA). Finaly, the cells were washed twice with PBS and analyzed.
Metabolic T~helinq with 35so~
COS cells transfected with expression plasmids enso~ ng mucin:immunoglobulin chimeras were trypsinized one day after transfection and transferred to new plates in complete medium (DMEM with 10% calf serum). Prior to labeling, the medium was removed, the cells were washed 35 once with PBS, and the medium was replaced w~th either W O 97/00079 PCTrUS96/10043 cysteine and methionine-free medium for labeling with [35S]cysteine and methionine (TrAn~TAhel, ICN) or with sulfate-free CRCN-30 medium (Sigma Chemical Co.) ~or lAheli~ with 35S04. Serum was not added, and 5 radionuclide was typically present at a co~centration of 200 ~LCi/ml. After a 1 ~hel i ng interval of 12 to 16 hour the supernatants were harvested, and the fusion proteins were collected by adsorption to goat anti-human IgG
agarose (Cappel). Adsorbed proteins were ~ubjected to 10 denaturing electrophoresis on 8% polyacrylamide gels under reducing conditions.
Chlorate Inhibition of Adhesion COS cells were transfected with D~F dextran and ~n~-lh~ted immediately in DMEM con~;n; ng 10% calf serum 15 and 10 mM sodium chlorate. One day after transfection the cells were trypsinized and ~nc~lhAted in fresh ~fih_~
in the ~ame medium for 6 hours. The medium was then removed, the cells were washed with PBS, and then inc~lh~ted for 18 additional hours in a custom prepared 20 DMEM medium (Life Technologie~) lacking sulfate and con~1 n i n~ 2% of the conventional levels of cysteine and methionine with 10% dialyzed fetal bovine serum in the pr6-Ance of 10 mM sodium chlorate (Baeuerle and Huttner, Biochem. Biophys. Res. Comm., 141:870-877, 1986). Cell~
25 were then harvested for use in the adhesion and immunofluore~Ac~rce assays. CollL~ol cells were treated similarly but were ~ncllh~ted in DMEM con~ning n~ yzed serum.
~-6Q Cell Rolling Video images of HL-60 cells rolling through a parallel plate rectangular flow chamber (FCS2, Bioptechs, Incorporated, Butler, PA) with a temperature controlled stage set at 37~C were acquired with an AIMS Technology (Bronx, NY) CCD camera mounted on a Zeiss ICM 405 35 inverted microscope equipped with a 2.5x objective. The W O 97/00079 PCTrUS96/10043 chamber height was 250 ~m. Cells were withdrawn through the chamber at a defined flow rate with the aid of a Harvard Apparatu~ (South Natick, MA) model I/W 22 syringe pump. Images were analyzed using NIH Image. To inhibt 5 ~ulfation, HL-60 cells were washed once with PBS and grown for 18 hours in sulfate free medium con~Ai~in~ 2%
of the normal levels of cysteine and methionine, 10 mN
codium chlorate, and dialyzed serum as deccribed above.
For each experiment, 106 cells were sus~n~ in 1 ml of 10 0.15 M NaCl, 3 mM CaC12 and drawn through the chamber.
Gla s coverslips were coated with affinity-purified goat anti-human IgG antibody at a ~o~Dntration of 10 ~g/ml in 50 mM Tri~-HCl (pH 9.0) for 2 hours, washed twice with PBS, and blocked overnight with 0.2% BSA in PBS. The 15 treated coverslips were then immersed in ~upernatants of COS cells transfected with the a~ iate i~munogloh~
chimers expression plasmids, washed twice with PBS, and assembled in the flow chamber.
The Ami no T~rminus of PS~T~ NecessarY for P-Selecti n 20 hin~l~n~
Deletions of the amino terminus of the PSGL-l mucin were created with PCR t~hn i ques, and the reeulting truncated cDNAs were inserted downstream of a secretory peptide ~equence which had been fused to a short 25 oligopeptide tag derived from influenza hemagglutinin (HA). Expression plasmids ~n~o~i n~ the truncated molecules (Fig. lA) were transfected into COS cells in the pre-~nc~ of a specific myeloid fucosyltransferase, designated FTVII, which directs the expression of ~LeX
30 determinants exclusively (S~ki et al., J. Biol. Chem., 269:14730-14737, 1994; Natsuka et al., [published erratum appears in J. Biol. Chem, 269:20806, 1994], J. Biol.
Chem., 269:16789-16794, 1994). Expression of the deletion mutants at the cell surface was confirmed by 35 indirect immunofluorer-~n~e using anti-HA monoclonal W O 97/00079 PCTrUS96/10043 ant~hoAies. The pr~~nc~ of sLeX on the cell surface wa~
similarly confirmed using the monoclonal antibody CSLEX-l. The ability of radiolabeled transfected cell~
to bind to plastic wells precoated with 5 P-selectin:immunoglohlll in fusion protein was determined.
These experiments revealed that deletion of the amino terminal 100 residues (referred to herein as the apical domain) of PSGL-l was sufficient to abolish bi n~ i ng of the transfectants to immobilized P-~electin (Fig. lB).
10 ~hese experiments also demonstrate that sLeX mediates P-selectin b;nA;n~, as expression of FTVII was required for P-se~ectin b;n~;n~ (Fig. lB; compare bar 2 with bar 3).
Ex~le~sion of the deletion variants at the cell surface was confirmed by indirect immunofluorer~~nce using anti-15 HA monoclonal ant;h~Aies, and the prer~n~e of ~LeX on thecell surface was confirmed using the monoclonal antibody CSLEX-l. Table 1 shows the mean f 1UOL ~ -~e~Ce intensity (MFI) of COS cells that were cotransfected with human ~lvllh and the deletion constructs (shown in Fig. lA), 20 and ~ubjected to indirect immunofluore~c~nc~ with antibody against the amino terminal flu peptide or sLeX.
Table 1 CQnstruct Ex~ression (MFI) Flu Slex PSGL-1-flu 5.0 37 Xbal 17.0 26 Xba3 20.0 28 Xba6 21.0 28 Xba9 12.0 26 Xbal2 6.0 30 Xbal6 6.0 25 W O 97/00079 PCT~US96/10043 Tn the Context Ql~ T.Arge, Sulfated Mucins~ the AminQ
Terminus of PS~T-l is Sufficient for P-Selec~-;n B;n~in~
To determine whether PSGL-1 seql~ncec other than those found in the first 100 N--ter ;nAl amino acid~
(i.e., the apical domain) of PSGL-l were required for bi nA; ng to P-selectin, the transmembrane and cytoplasmic regions of PSGL - 1 were replaced with those of the CD43 antigen (Pallant et al., Proc. Natl. Acad. Sci., 86:1328-1332, 1989; Shelley et al., Proc. Natl. Acad. Sci., 10 86:2819-2823, 1989). The refiulting molecule, which did not contain cysteine residues, holln~ P-selectin with the same efficiency as PSGL-l did (Fig. 2A and Fig. 2B).
Thus, neither disulfide bond formation nor a specific ~embrane An~oring segment is required for P-selectin 15 b; n~ ~ ~ activity.
The predicted first lOo amino acids o~ PSGL-l were then genetically grafted onto the amino termini of mucin-like repeat elementc of several unrelated mucins to determine whether or not ~he PSGL-l apical domain is 20 sufficient for P-selectin ligand (i.e., counterreceptor) activity (Fig. 3A). Certain of these chimeric mucins were able to ~u~o-L P-selectin bin~in~ in this setting.
CD34 and CD43, two relatively large mucins found predominantly on human hematopoietic cells, were both 25 able to ~u~LL bin~in~ In ~o,.L ast, an artificially ~n~h~red variant of GlyCAM-1, a mucin expressed on high endothelial venules that has L-selectin ligand activity (Lasky et al., Science, 258:964-969, 1992), was inactive in this assay (Fig. 3B). The GlyCAM-1 ~ucin domain in 30 these experiments was tethered to the cell surface via the extracellular stalk, transmembrane ~ i n ~ and cytoplasmic Anchoring segments of CD7 (Aruffo et al., EMBO J., 6:3313-3316, 1987). Cell surface expression of the different mucins and mucin chimeras was confirmed by 35 indirect immunofluor~rc~ using an~ihoAies agains flu W O 97/00079 PCTAUS9~10043 tag, sLe~, or the ~e_~e-tive mucins. Table 2 shows mean fluoreccence intensity (MFI) measurements of expression of flu tag or sLeX by COS cells transfected with the constructs analyzed in Fig. 3B. CD34 and CD43 construc~s 5 were positive for expression by indirect immunofluorescence using cognate anti-CD ant~ho~te T~hle ~
Construct ~xl~e-s~on (J~ T) 10 ~ --- 52 PSGL-1-flu 9.8 43 PSGL-l-NH2/CD43 rep. 12 67 CD34 --_ 50 PSGL--l--NH2/CD34--COOH 8.0 33 Glycam-flu 12 32 PSGL--l--NH2/Glycam--COOH 8.0 28 The apparent mol~clllAr masses of CD43 and CD34 expressed in COS cells are Le~G~Led to be 100-130 kD
(Shelley et al., Proc. Natl. Acad. Sci., 86:2819-2823, 1989) and 100 kD (Simmons et al., J. Immunol., 148:267-271, 1992), respectively; the PSGL-1 monomer exhibits an effective mol~c~ ~ mass of 110 kD (Sako et al., Cell, 75:1179-1186, 1993). GlyCAM-1, in its native (untethered) 2S state comigrates with 50 kD proteins, suggesting that it i8 substantially ~;maller (Lasky et al., Science, 258:964-969, 1992). In our studies, the larger mucins were able to ~u~po~L P-selectin b; n~; ng when the apical domain of PSGL-1 was appen~ to the amino terminus of the mucins.

Sequential deletion of the internal repeat elements of PSGL-1 allowed us to shorten the molecule in a systematic manner without compromising potential global tertiary a~sociations (Fig. 4A). As the~e repeat elements were 5 delet~d, the bin~;ng activity of PSGL-1 declined, consi~tent with the conclusion that distance from the plasma membrane is an important determinant of P-selectin bin~in~ activity (Fig. 4B).
Our data also indicate that sulfation i8 one 10 determinant of the ability of ~ucins to ~ o-~ apical domain-directed bi nA;ng. We AÇ:r~ the ability of vari~us ~ucins to undergo sulfation in COS cells.
PSGL-l~ CD34, CD43, and GlyCAM-1 soluble mucin chimeras readily inco~ ated sodium 35S-sulfate when ex~ in 15 COS cells (Fig. 5).
Tn~h;tion of Sulfation Blocks PSGL-1 Bjndin~ to P-Select~ n We have found that inhibition of sulfation blocks PSGL-l bin~ing to P-selectin. COS cells were 20 cotransfected with PSGL-l and FTVII, or transfected with PSGL-l and FTVII separately. During the time period in which maximum synthesis of PSGL-l was expected, the cells were inr~hAted in a modified DMEM medium lacking sulfate and cont~i n ~ ng 10 mM sodium chlorate, a relativelly 25 selective inhibitor of sulfation (NaC103). We observed a significant decrease in the ability of chlorate-treated cotransfected cells to bind to immobilized P-selectin (Fig. 6A), whereas the same cells showed little or no decrement in b~r~A~ ng to immobilized 30 E-selectin (Fig. 6B). Cell surface expression of either the sLeX antigen and the PSGL-l amino terminal tag sequence was not inhibited by NaC103 treatment. In fact, as shown in Table 3, an increase in the mean fluorec~nce intensity of the transfected cells, representing both 35 anti-sLeX and anti-flu tag, was observed following CA 0222462~ 1997-12-12 W O 97/00079 PCTrUS96/10043 chlorate treatment, suggesting that chlorate may affect inter~Al~7~tion or cell surface export.
Table 3 Expression (MFI) w/o N~C103 W/10 m~ N~C103 sLex Flu sLex Flu ~ ln 23 --- 30 --PSGL-l-flu --- 9 --- 22 PSGL-lflu + ~L~llh15 10 35 34 A soluble PSGL-1 im oglobulin chimera synthesized under comparable conditions showed 10 essentially complete inhibition of 35s-sulfate in~r~ation (Fig. 7), under conditions in which prote~n synthesis as measured by [35S]cysteine and methionine inco~Lation was not inhibited. These data demG.lDL~ate that sulfation of the P-selectin ligand is required for 15 P-selectin b1 n~ i ng activity.
Fin~ Structure Deletion Analvsis o~ the A~ical Domain of PSt'-T~--l To loç~li7e the elements within the 100 amino acid apical domain which contribute to P-selectin ligand 20 activity, we prepared a collection of deletion mutants in which various regions of the apical domain were deleted (Fig. 8A). Each amino terminal deletion mutant was then placed downstream of the CD5 leader/flu tag element to monitor cell surface expression. The fine structure 25 deletion mutants showed little variability in their ability to express the epitope tag, as ~ by indirect immunofluorescence. Removal of the first 20 amino acids of the N-terminus of the mature PSGL-l did not affect P-selectin bin~;nq activity. In ooIlL~ast, 30 removal of the first 40 amino acids of the N-terminus abrogated bi nA i ng (Fig. 8B). Further deletions of PSGL
did not affect P-selectin b;n~ing activity. Accordingly, W O 97/00079 PCT~US96/10043 amino acid residues 20 to 40 of PSGL (i.e., residues 38 to 57 of the predicted precursor having the signal seguence) are required for P-selectin b;n~;nq.
To demonstrate that residues 38 to 57 are 5 sufficient for PSGL-l apical domain-directed activity, we Arp~nA~ this segment to the amino termini of PSGL-1 and CD43 mucin cores from which the apical domains had been deleted (Fig. 9A). In both cases, addition of amino acids 38-57 of PSGL-l peptide element conferred P-selectin 10 bi n~ i n~ activity upon the mucin core. In both cases, the level of P-selectin bin~i ng activity was equivalent to that Pf native PSGL-l (Fig. 9B).
Specific Residues With;n the Amino Termin~l Pe~ de Are Re~li~ed for P-selectin Bin~;ng Activ;ty The 20 amino acid region which i8 n~ce~ ~y for P-~electin binAin~ contains three potential tyrosinQ
~ulfation sitQs and two thr~on;n~ residues for 0-l;nk~
gly~ylation. To A ~-~ C the importance of these rQsidues, the tyrosines were converted to phenylAlAninQ
(Fig. 9A). In a ~ecQ~A peptide, the thr~on;n-- werQ
converted to Al An; n~fi~ In addition, a third peptide, contA; n; ng a quintuple mutation, was prepared such that both conver~ions were made in a single peptide. Each mutated peptide was then positioned, separately, 25 downstream of the flu tag and u~L.eam of either (1) the truncated PSGL-l lA~k;ng the apical ~ ~;n~ or (2) the CD43 repeat elements and transmembrane domain. Cells expressing the resulting chimeras were tested for their ability to bind to immobilized P-selectin (Fig. 9A).
30 Conversion of the tyrosines to phenylAlAn;r~- resulted in a loss of binding activity to P-selectin. Replacement of the threonine residues with Al~ n i n~ dimi n; ~he~ b; n~; ~g ~
but did not abolish it entirely. Expression of the flu tag or sLeX epitope was not affected in these cells.
35 Binding -~;Ated by the apical 20 esidues was, like that W O 97/00079 PCT~US96/10043 - a4 -of native PSGL-l, ~p~n~nt on the pr~en~e of calcium.
These data indicate that sulfation of tyrosines at positions 46, 48, and 51 is required for P-selectin hin~inq activity. E-selectin bin~ing was unaff~cted S under the same condition. In addition, these data indicate that the thr~Qrl;n~- at position~3 44 and 57 are required. These thr~onin? residues can serve as sites for o-l ink~ glycan addition. These experiments, in con~unction with our experiments showing that FTVII
10 e~Le~sion is n?ce~A~y for P-selectin bin~inq~ provide cv;~nce that P-selectin b; n~;nq requires sLeX at thr~?o~in~3 44 and 57. In sum, the above--described experiments demonstrate that amino acids 38-57, contA; ni~q three residues for sulfation and two residues 15 for sLeX addition, are sufficient to confer P-selectin bi n~nq activity.
Residues Within the Amino-Terminal 20 Amino Acids ~e Slllfated on TYrosine To determine whether the amino-terminal segment 20 was capable of being sulfated in vivo, we created fusion proteins consisting of the native or mutant peptide 5e~l~n~e~ ~oined to human immunoglobulin Gl (IgG1) (Fig.
12A). The resulting fusion proteins were e~ in COS cells, and their ability to assimilate inorganic 25 sulfate was A~cecrr~ (Fig. 12B). Immunogloh~ n chimeras bearing the native peptide se~l~nc~c were capable of incorporating sulfate, whereas those bearing phenyl~lAnine substituted for tyrosine were not (Fig.
12C). Replacement of threonine with AlAnin~ had no 30 effect on ulfate incorporation (Fig. 12C).
Tnh;hitors of Sl~lfation Block ~T.--I;0Rollina on P--Select;n-T~unoqlobulin Chimeras To explore whether inhibition of sulfation would compromise a physiologically relevant adhesion, we 35 subjected HL-60 cells to growth in medium contAining chlorate and examined the ability of the reculting cells to attach and roll on coverslips coated with P-selectin-i unogloh - l i n chimeras under conditions of defined fluid shear ~L~ (Lawrence et al., Blood, 75:227-237, 1990).
5 HL-60 cells were capable of attaching to and rolling upon coverslips precoated with P-selectin-immunoglQhllin chimeras, whereas no such interaction was observed with lips coated with a cD4-immunoglQh~ n chimera (Fig.
13). Growth of HL-60 cells in chlorate dramatically 10 r~llce~ the frequency of cell interaction with the substrate (Fig. 13).
An~ih-~dies ;-ntl Ant;hody Fusion Proteins Beari~c7 Sia T,~X Antl Sl~lfAt~rl Determinilnts In one emhoA;ment, the invention features an 15 antibody bearing sialyl-LeX and sulfated determinants.
Such an anti~ody may be created by ill~r~ducing sulfation sit2s ti.e.~ a tyrosine in an acidic context) into an existing antibody molecule in the vicinity of an il~ ollc~A or existing sialyl-LeX addition site (for 20 example, by s~AnAA~d site-directed mutagenesis).
Alternatively, a~o~iate sialyl-LeX and/or sulfation site~ may be added by Arr~A i n~ any P-selectin ligand ~equence (for example, any P-selQctin domain dQscribQd herein) to a naturally-occurring antibody sequence (for 25 example, IgG or IgM) by stAnAA~d recombinant DNA
~chn i que8 to produce a P-selectin-antibody fusion prot~in. Preferably, the P-selQctin ligand ssquQnce is Arr~A~A to the amino-terminus of the antibody molecule.
Such an~ihoAies are useful for disrupting undesirable 30 interactions between cells or proteins, or, generally, for disrupting any interaction between two molecules, one of which bears a determinant carried by the antibody.
Because thQse determinants normally act to facilitate interactions involving E seleatin and P-selectin (e.g., 35 interactions between neutrophils and endothelial cells . CA 0222462~ 1997-12-12 PCT/US 96 /1 ~ 043 IP~A/US ~ 4 J A N 1997 -_ - 26 -lining the blood vessel walls), the abllity to disrupt such interactions provides many therapeutic applications, for example, in minimizing inflammation and decreasing extravasation-dependent organ damage and/or clotting.
In addition, if desired, one or more sialyl-LeX
moieties which mask the CH2 portion of the immunoglobulin molecule and thus inhibit complement fixation and Fc receptor binding may also be incorporated into the antibody sequence. Because the carbohydrate moieties 10 block the immunoglobulin domain which triggers complement fixation and Fc receptor binding, such antibodies do not '~, elicit the undesirable side effects (i.e., those resulting from complement ~ixation and Fc receptor binding) frequently associated with antibody-based 15 therapies. Preferably, the carbohydrate groups serve not only to inhibit undesirable complement fixation and Fc receptor binding, but also perform the function of competitively inhibiting an E-selectin and/or P-selectin mediated intracellular interaction.
To inhibit complement fixa~ion and Fc receptor binding, sialyl-LeX determinants may be added to the antibody molecule at any appropriate site. N-linked glycan addition sites are well known to be: N X S/T
(where N is asparagine, S is serine, T is threonine, and 25 X is any amino acid except proline). Accordingly, an exemplary molecule may be designed that includes several such sites for attachment of sialyl-LeX side chains.
Inspection of the IgG1 sequence (Fig. lOA-E) reveals at least five sites at which N-linked glycan addition sites 30 may be introduced into the molecule in advantageous locations, where complement ~ixing and Fc receptor binding ability will be impaired by the process. These sites include amino acid residues 274, 287, 295, 322, and 335.
Although these are preferred sites of N-linked glycan 35 addition, they are not the only candidates; other AMENDED S~lEET

u3eful sites may be identified and in~-~v~ated into the IgGl sequence using, a~ ~l;~Ance~ the following criteria:
(1) the sites are, preferably, located in the CH2 region of the immunoglohlll ;n molecule, i.e., in the portion of 5 the molecule responsible for complement fixation and Fc receptor b; nA; n~; (2) the sites are located in regions of the sequence, predicted by their hydrophilic nature, to be pre~ent on the outside of the immunoglobulin molecule and therefore A~ce~cible to the enzymes r~cpQ~ible for 10 attachment of carbohydrate side rhA;n~; (3) the sites are located in a region which is minimally disruptive to the prim~ry Amino acid sequence and, thus, the predicted ~e~-onAAry amino acid structure. For example, a naturally-occurring site which differs from an N-l~nkeA
15 glycan addition site by a single amino acid would be preferable to a site requiring two alterations in the amino acid sequence. Moreover, it is preferable to create an N-linked glycan addition site by substituting amino acids of similar charge or polarity (e.g., 20 substitution of one l~n~hArged amino acid for another).
One or more N-li nk~A glycan addition site substitutions may be engineered into a particular IgGl-~ncoA i ng seguence; such sequences (i.e., those which ~nroA~ an antibody molecule to which sialyl-LeX moieties are 25 attA~h~A) are termed IgG1-sialyl-LeX or IgGl-LeX.
The il-L~duction of additional glycosylation sites at amino acids ~274, ~287, and #322 within the C~2 domain created a molecule that was unr~cogni~ed by Fc receptor or complement using assays that are st~n~Ard in the art;
30 exemplary co~plement fixation assays include Weir et al., ~nAhook of Experimental Immunology, Blackwell, Oxford;
and Coligan et al. Current Protocols In Immunology, Wiley Interscience, 1995.
A particular IgGl molecule bearing sialyl-LeX
35 moieties ic proA~lre~ as follows. The IgGl gene is . CA 0222462~ 1997-12-12 PCTi1J~ ~6 /1 0 043 IP~ ; 1 4 J A N 1997 publically available, and its sequence is shown in Fig.
10. The gene is mutagenlzed by s~andard methods of in vitro site-dlrected mutagenesis in order to introduce one or more N-linked glycan addition sites (e.g., those 5 described above and shown above the naturally-occurrlng sequence in Fig. lOA-E). The gene is then inserted into a vector designed to express the protein in a eukaryotic cell (see, e.g., those vectors described ln Glllles et al., U.S. Patent No. 4,663,281, hereby incorporated by 10 reference). The eukaryotic host cell is preferably a m~mm~l lan cell (e.g., a CHO or lecll cell), and the expression vector containing the mutated IgGl-LeX-encoding sequence is introduced into the hoqt cell by transient or stable transfection using standard techniques. Such host 15 cells are also transfected (translently or stably) with a vector capable of expressing an ~(1,3)fucosyltransferase capable of attaching the slalyl-LeX groups to the antibody molecule at the glycosylation sites. The ~(1,3)fucosyltransferase gene may be expressed from a 20 vector dlstinct ~rom that encoding IgGl-LeX, or both genes may be carried on, and expressed from, a common vector.
~mm~l ian cells are particularly useful hosts for the synthesis of IgGl-LeX because they provide all required precursors for slalyl-LeX production.
To produce the sialyl-LeX-modified and sulfated antibodies of the invention, the gene encoding the antibody sequence is preferably expressed in a cell which also expresses an ~(1,3)fucosyltransferase that exclusively catalyzes ~(1,3)fucose linkages; such an 30 enzyme is described in Walz et al., Science 250:1132-1135 (1990) and in Seed, U.S.S.N. 08/483,151, entitled "Fucosyltransferase Genes and Uses Thereof," filed June 7, 1995 (hereby incorporated by reference). Less preferab y, the ~(1,3)fucosyltransferase cDNA described AMEND~D SHEET

W O 97/00079 PCTrUS96/10043 in Lowe et al. (Cell ~:475, 1990) may be utilized. This fucosyltransferase re~o~n;7es a sialylated precursor molecule and adds either an ~(1,3)- or an ~(1,4)-link~A
fucose moiety to N-acetylgll~o~mine side ~h~ i n~ . The 5 sialyl-Le~ determinant is characterized by an ~(1,3)-l~n~A~Q, and, as such, the ~(1,3)fucosyltrans~erase enzyme of Lowe (~u~r~) proA-lr~ both the desired sialyl-LeX-modified molecules and products bearing ~gl,4)-l;n~e~
fucose which, al~ho~yh not active in bin~ to P-10 selectin and E-selectin, do not interfere with the action of the sialyl-Le~-modified molecules nor produce other unde~irable side effects.
Host cells expres~ing ~(1,3)fucosyltransferase and the antibody to be modified are grown by 8t~n~d 15 methodfi, and the antibody i8 purified from a cell lysate hAr~~ on its affinity for a Protein A column or any other 8~n~d te~hn;que of antibody isolation and purification.
~l-Acid Glyco~-o~ein-Antibody Fusion Proteins Bearing 20 Sialyl-LeX and Sulfated Determinants As ~c~ herein, antibody fusion proteins modifi~d by sulfation and sialyl-LeX addition have importAnt therapeutic and diagnostic uses. Previous work has demo~.~L~ ated that large amounts of antibody fusion 25 proteins may be generated and secreted transiently from tran~fected mammalian cells (for example, COS cells). In general, to produce an AGP antibody fusion protein accor~ing to the invention, DNA ~nco~; ng an AGP and a P-selectin ligand domain are fused in-frame to human IgG
30 domains (for example, constant domains) by st~n~d techn;ques, and the fusion protein is expressed, also by s~A~d ~echn;ques. The antibody portion of the molecule facilitates fusion protein purification and also prolongs the plasma half-life of otherwise short-lived 35 polypeptides or polypeptide domains. Preferably, . CA 0222462~ 1997-12-12 PCTIU~96/~04~
IPEA/IIs 1 4 J A N 1997 _ - 30 -antibody fusion proteins are expressed according to the methods disclosed in Seed et al., U.S.S.N. 08/483,151 entitled "Fucosyltransferase Genes and Uses Thereof,"
filed June 7, 1995 (which is hereby incorporated by 5 reference), e.g., using IgG or IgM antibodies or portions thereof (see also Zettlemeisl et al., DNA Cell Biol.
9:347 (1990) for IgM fusion proteins).
Recombinant plasmids expressing particular AGP-antibody fusion proteins (e.g., AGP-Hinge-CH2-CH3 and 10 AGP-CH2-CH3 proteins) have been constructed as follows.
A cDNA encoding the acute phase ~l-AGP gene was cloned from a human liver cDNA library by polymerase chain reaction (PCR) using oligonucleotide primers corresponding to the 5' and 3' coding regions of ~1-AGP
(Board et al., Gene 44:127, 1986) according to standard techniques. The 5' AGP primer was designed to contain a HindIII restriction site and the 3' primer was designed to contain a BamHI restriction site rather than the AGP
stop codon. The PCR-amplified product was digested with 20 HindIII/BamHI and cloned into a HindIII/BamHI-cut plasmid expression cassette (see Aruffo et al., Cell, 61:1303, 1990) containing constant domains of human IgG1 (i.e., Hinge-CH2-CH3 or CH2-CH3). A nucleotide sequence and amino acid sequence of this AGP-IgG fusion protein are 25 shown in Figs. llA-B and Fig. llC, respectively.
To create a molecule that blocks P-selectin-mediated interactions, sites for sulfation and, if necessary, sialyl-LeX addition are introduced into the antibody fusion protein sequence (for example, the 30 antibody fusion proteins described above). Such sites may be incorporated into an existing fusion molecule, for example, by introducing one or more sulfation sites (i.e., a tyrosine in an acidic context) in the vicinity of an introduced or existing sialyl-LeX addition site (for 35 example, by standard techniques of site-directed AMENDED SHEET

mutagenesis), or a P-selectin ligand sequence (for example, any of the P-seleetin ligand se~l~ne~c deseribed herein) may be Arren~ to the antibody fusion protein sequence using s~n~A~d ~ec~n~ques of reeombinant DNA
5 te~n~logy.
The P-selectin-AGP-antibody fusion genes are then il.L~ e~ into expression plasmids, and the plasmids are transfeeted into any a~ iate fucosyltransferase-~x~ ing cell for the production of soluble antibody 10 fu8ion protein~.
To prepare an antibody fusion protein eapable of ~nh~h~ting eomplement fixation and Fc reeeptor bin~ng~
additi~nAl 5ialyl-LeX rQn~nCllc gly~ylation sites (N-X-T/S) may be i.~L~ol.l~eA into the CH2 domain of human IgGl 15 a8 described above.
RAf-~'1 on this eonE;truction strategy, any number of reeombinant P-selQctin-AGP-antibody fusion protQins may be designed having long plasma half-lives and the ability to inhibit undesirable eell-eell interaetions (for 20 Qxample, the interactions between leukocytes and selectin-bearing cells). To generate molecules with heightened inhibitory potency, c~n~ te molec~ are de~igned and screened using the assays described above.
In one particular example, molecules may be screened for 25 their ability to incorporate sialyl-LeX and ~ulfated aeterminants and block the b;n~;ng of ne~Llo~hils to activated endothelial cells; such molecules find use in the inhibition of ~electin-~ren~nt inflammatory reactions and tissue injury inflicted by invading 30 leukocytes.
Molecl~les Ca~able of Interferinq with P-Selectin-Mediated An~ E-Selectin-Mediated Interactions Because both P-selectin- and E-selectin-mediated intracellular interactions are involved in inflammation 35 and heCs~ ? the crucial determinants involved in those CA 0222462~ 1997-12-12 W O 97/00079 PCTrUS96/10043 interactions have now been idQntified, it i~ possible to de~ign a single molecule capable of interfering with both typQs of deleterious interactions. In particular, molQculQs (for example, proteins) may be c~l.~L ~cted that 5 include both a P-selectin ligand domain (i.e., a domain bearing sialyl-LeX and sulfated moieties) and an E-~electin ligand ~ ~ ; n ( i . ~ ., a domain bearing a sialyl-LeX moie~y). Such a molecule may be constructed by combining domains, for example, by A~r~n~ing a P-select~n 10 ligand domain to a sialylated molecule (for example, a sialylated antibody or antibody fusion protein described hereLn). Alternatively, a~Lop~iate sialyl-LeX and/or ~ulfation sites may be illL.~llce~ into an existing sequence, for example, by site directed mutagenesis.
Gly~o_ylation or sulfation of an engineered molecule may be tested, for example, as described herein and in Walz et al., Science 250:1132-1135 (1990). The ability of a ~ialyl-LeX-modified and/or sulfated molecule to interfere with intracellular interactions may also be 20 tested as de~cribed in Walz et al., supra, or by any 5~n~d techn;~ue, for example, by assaying the ability of increasing concentrations of the determinant-bearing molecule to i~hibit adherence of T lymphocytes or ~yelo~d cells to immobilized P-selectin and/or E-selectin.
25 Use For administering a protein or organic molecule of the invention to a patient, the pharmaceutically-pure protein or molecule is suspe~P~ in an acceptable carrier, e.g., physiological ~l in~, and is delivered to 30 the patient by any appropriate route (for example, intravenously) in a single dose or in multiple doses.
Optimally, a sufficient guantity of the therapeutic is provided to saturate all P-selectin and, for a dual function molecule, all E-selectin b;n~;ng sites on an 35 endothelial cell. Typically, thi~ may be achieved with W O 97/00079 PCT~US96/10043 ~ 33 ~
doses of 0.1 mg/kg or greater. The preferred dosage is in the range of 0.1-2.0 mg/kg.
The sialyl-LeX-modified and sulfated molecules and proteins of the invention (for example, the modified 5 an~iho~ies and antibody fusion proteins described herein) may be used, in one example, for the treatment of extravasation-A~r~n~ent organ damage and/or clotting. In particular, heC~l~re r --1 ectin mediates the attachment of n~LL-J~ overlying ~ites of inflammation or ~ 7~
10 damage or proximate to thrombus formation, the molecules and protein~ of the invention provide useful therapeutics for bl~ n~ such interactions. For example, P-selectin likely mediates the migration of neutrophils into the lung following adult respiratory distress syndrome and 15 into the heart following ~ h~mic myocardial injury (i.e., infarction), and may play a role in glomerular damage to the k;~ln~y8 under certain condition~.
Accordingly, a sialyl-LeX modified and sulfated mole or protein of the invention may be administered to a 20 patient suffering from such a ~ir~e or condition. Such treatment attenuates extravasation-A~ren~nt damage by competitively inhibiting the interaction between the invading ne~ro~hils and the endothelial cell of the blood vessel or organ. The comrolln~c of the invention, 25 particularly, P-selectin ligand-AGP fusion proteins and P-~eleatin ligand-AGP-antibody fusion proteinc may also be used, as described above, for the treatment of septic shock or septicemia.
In addition, an~iho~ies or antibody fusion 30 proteins according to the invention may be used in conventional ~erhn iques of antibody-based therapies or in v vo diagnostics, ~Aking advantage of the antibody's specificity to target therapeutic or diagnostic sites.
In one particular example, the P-selectin ligand domain 35 of an antibody fusion protein acc~-ding to the invention CA 0222462~ 1997-12-12 W O 97tO0079 PCT~US96/10043 targets that protein to a site of inflammation and provides both a therapeutic (useful for blor~i n~
deleterious P-selectin-mediated intracellular interactions) and a ~;Agn~ctic (useful for tagging the 5 site of inflammation). Agstin, attA~he~ ~ialyl-LeX
determinants may be used to mask the CH2 domain of the antibody and block the undesirable effects of complemen~
fixation and Fc receptor b;n~in~.

Other ~odiments Other QmbodimQnts are within the claims. For ~xample, for the yu~o~e of blocking interactions between cQlls or proteins, any other ap~lop~iate carrier molecule to which a sialyl-LeX and a ~ulfated determinant may be attachad may be utilized in the invention. Generally, 15 proteins are preferred h~CAl~e of their relatively long half-lives in serum. One class of carrier proteins are serum proteins such as albumin (e.g., bovine serum albumin or human serum albumin), transferrin, or ~-2 macroglobulin. The carrier proteins may contain 20 endogenous sulfation and glycan addition sitQs in addition to which site~ are i"Llollc~ into the DNA
seguence of the carrier protein (as described above) byp for example, site-directed mutagenesis. The carrier molecule, less preferably, may be a lipid. In one 25 example, the lipid, with one or more attA~h~ sialyl-LeX
and sulfated determinants is delivered as a liposome to a target cell wall (e.g., an endothelial cell wall). The l~rQ~ome may block a cell or protein interaction or may be used to deliver a drug to its a~ iate ~ite of 30 action.
Production of carrier molecules bearing sialyl-LeX
and sulfated determinants may be carried out in a cell, preferably, a eukaryotic cell other than yeast.
Nammalian cells, e.g., mammalian cell lines, provide W O 97/00079 PCTrUS96/10043 particularly suitable hosts. These cellc generally synthe~ize the n~aeFCAry precursor molecules and produce or can be engineered to produce the enzymes ~~Gl.sible for sulfation and carbohydrate attachment. For the 5 attachment of sialyl-~e~ determinants, mammalian cell lines ~uch as CH0 and lecll are particularly ~uitable.
Alternatively, either or both of the sialyl-LeX and sulfated determinants may be attA~hPA to a carrier molecule in vitro, i.e., extracellularly. In one 10 example, ~(1,3)fucosyltransferase would be bound to a solid ~u~p~LL (e.g., a column) and a sulfated carrier molec~le pA~~~ over the ho~n~ fucosyltransferase enzyme, under conditions which facilitate attachment of sialyl-LeX y-O~y~ to their a~Liate site(s) on the carrier 15 molecule.
The invention also encompAsses the use of sulfated and sialyl-Le~-modified AGP-antibody fusion proteins for protecting against, inhibiting, or treating a shock-inducing event, the clinical manifestations of '-~9C!l', or 20 both which are caused by microbial factors (e.g., opolycAc~hArides (LPS)), microbial toyinc (e.g., toxic ~oc~ enterotoYinc)~ host mediators (e.g., cytQk~n ), or anti-tumor therapies (e.g., administration of tumor necrosis factor (TNF) or interleukin-l (IL-l)), or any 25 combination thereof. For example, such an antibody fusion protein can be administered to a human patient to alleviate the effects of septic shock inAllceA by microbial LPS. The ability of an antibody fusion protein to protect against, treat, or inhibit the effects of 30 ~hock (e.g., septicemia or toxic shock syndrome) is evaluated according to st~nA~rd methods known in the art (e.g., those described in Libert et al. (1994) ~. Exp.
M~d. 180: 1571-1575).
All publications, patents, and patent applications 35 mentioned in thi_ specification are herein in~L~o~ated W O 97/00079 PCT~US96/10043 by reference to the same extent as if each individual publication, patent, and patent application was specifically and indiv~ Ally indicated to be in~GLated by reference.

W O 97/00079 PCTrUS96/10043 SEQUEN OE LISTING
(1) GENERaL INFORMATIONs (i) APPLICANT: The General Hospital C~.~G.aLion (ii) TITLE OF lhv~-lON: P-SELECTIN LIGANDS AND ~TAT~n ~T~C~T~S
AND ~T~O~S
(iii) NUMBER OF SEQU~N OE S: 14 (~v) ~ rONDEN OE An~SSs ~A~I AnD~ Y~s Fish & Richardson P C
,BI STREET: 225 Franklin Street C, CITYs Boston D STATB: MA
E~ ~UUh ~: USA
~F, ZIP: 02210-2804 (v) OOMPUTBR ~n~RT~ FORM:
IAJ M~DIUM TYPE: Floppy disk B CX~u.~n: IBM PC c~ ,-';hle ,C OPERATING SYSTEM: PC-DOS/NS-DOS
~DJ SOFTWARE: P~ten~Tn n~le~e ~1.0, Version ~1 30 (vii) PRIOR APPLI Q TION DATAs (A) APPLI Q TION Nur~R~s US 60/000,Z13 ~B) FILING DATEs 14-JUN-1995 (C) CLASSIFICATIONs (viii) A~.~nN~/AGENT lN~Ok~ATIONs (A) NAMEs Lech, Raren F.
(B) REGISTRATION NUMBBRs (C) n~Y~N OE /DOCKET r~rUR~s 00786/28WOl (iX) T~T~ rlcATIoN lN~ OndATION:
(A) TELEPHONE: 617/542-5070 (B) TELEFAXs 617/542-8906 (C) TELEYs 200154 (2) lN~O~ATION FOR SEQ ID NOsls (i) SEQU~N OE CHARACTERISTICSs A LENGTH: 10 ~mLno acid~
~B TYPE: amino acid C, sT~Nn~n~Ss not relevant ~DJ TOPOLOGY: lin~ar (ii) Y~r~CnT~ TYPEs prot~n (xi) SEQUENCE D~CC~TPTION: SEQ ID NO:l:

Ala Thr Glu Ala Gln Thr Thr Pro Pro Ala (2) lh~O_~ATION FOR SEQ ID NOs2s CA 02224625 l997-l2-l2 W O 97/00079 PCT~US96/10043 ~i) 8EQU~N OE ~TPRTSTICSs IAJ LENGTHs 18 amino acids rBJ TYPES amino acid rC, STR~N~-F.~r-~-CSS not rel~vant ~DJ TOPOLOGYs linear (ii) MnT-~C~T-T~ TYPEs protein (xi) SEQUEN OE P~RTPTIONs SEQ ID NOs2s Met Ala Thr Asn Ser Leu Glu Thr Ser Thr Gly Thr Ser Gly Pro Pro Val Thr (2) INFOPMATION FOR SEQ TD NOs3s (i) SEQUEN OE ~AR~rT~RT~TICSs (A'l LENGTHs 42 amino ncid~
Bl TYPEs amino acid .,C, STR~N~ cs: not rele~ant ~D~ TOPOLOGY: linear (ii) M~T~CnT~ TYPEs prot~in (xi) SEQUEN OE D~-qCRTPTION: SEQ ID NOs3s Gln Leu Trp A~p Thr Trp Ala A~p Clu Ala Glu Ly~ Ala LRU Gly Pro Leu LQU Ala Arg A~p Arg Arg Gln Ala Thr Glu Tyr Glu Tyr L~u Assp Tyr A~p Phe Leu Pro Glu Thr Glu Pro Pro (2) ~rORiLATION FOR SEQ ID NOs4s (i) SEQUBNCE ~R~T~RTCTICSs rA~ L~NGTHs 20 amino acid~
B TYPBs amino acid C, S~R~N~ CSs not rel~vant ~DJ TOPOLOGYs linHar ( ii ) M~T-~C~T ~ TYPBs prot~in (xi) SEQUEN OE p~C~RTPTIONs SEQ ID NOs4s Arg Anp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr LQU Asp Tyr Asp Phe LQU Pro Glu Thr (2) lNr~K~ATION FOR SEQ ID NOs5s (i) SEQUENOE ~R~T~PTSTICSs CA 02224625 l997-l2-l2 W O 97/00079 P ~ fiUS96~0~43 ~Aj T- _~S 20 amino acid~
~B TYPE: amino acid 'C STRP~ Ss not ralevant ~D~ TODOLOGYs linear (ii) ~T~Cn~-R TYPEs protoin (xi) SEQUENCE ~CC~TPTION: SEQ ID NO:5:
Arg A~p Arq Ar~ Gln Ala Thr Glu Ph~ Glu PhQ Leu A~p PhQ A~p Ph~

~ou Pro Glu Thr (2) lN ~RMATION FOR SEQ ID NO:6:
(i) SEQUEN OE r~R~DT~TICSs ~A~l LENGTHs 20 amino acid~
BI TYPBs amino acid C, S~RP~ ASs not r~levant DJ TOPOLOGY: linear ( ii ) ~nT~CnT-~ TYPE: protQin (xi) SEQUEN OE ~ACRTPTION SEQ ID NOs6:
Arg Asp Arg Arg Gln Ala Ala Glu Tyr G u Tyr Leu Asp Tyr A p Phe L~u Pro Glu Ala (2) INFOR~TION FOR SEQ ID NOs7s (i) SBQUEN OE r~R~rT~RTRTICSs ~AJ LENGTHs 20 amino acid~
IB TYPEs iino acid ,C, STD~r~ -CSs not r~lovant ~DJ TOPOLOGYs linear ii ) ~r-~C~n-~ TYPE: protein (xi) SEQUENCE D~-~RTPTION: SEQ ID NOs7:
Arg A~p Arg Arg Gln Ala Ala Glu Phe Glu Phe Leu A~p Phe A~p Phe Leu Pro Glu Ala (2) lN~OR~ATION FOR SEQ ID NOs8:

W O 97/00079 PCTrUS96/10043 (i) ~~QuENcB ~RaRa~T~T.CTICS
~A, LENGTH~ 2287 ba~Q pair~
BI TYPEs ~l~i~ acid ~C ST~ ASS ~ingl~
,DJ TOPOLOGY: lin~r (ii) MnTRC~TR TYPB: DNA (~
(xi) SlSQUll:NOE l:!~CCPTPTIONs SE:Q ID NO~8s AAGCTTACCA CCATGGACTG GACCTGGAGG ~~C~ .,v.Gv.GGC ~~~r~CT~A~ 60 Gv.v.C~AGT CCCAGGTGCA G~vv~v~AG -,~,GGGv~,G AGGTr-aar-aa GC~-G~v.~C 120 ~Gv~vAAGG -~ ~AA GG~,,~-,vvA GC~~~CTTCA Gc~cTaTGc TATCAGCTCG 180 GTGC~~~ CC~-~GvACA AGGG~..vAG TGGATGGGAG GGAT Q TCCC TA-, vv- 240 rA~A~~T rA~ar~ GTTcr~y~Gc AGAGT Q CGA T~ArCGCGr-a CGAATC QCG 300 Ar~ ccT A QTGGAGCT G~ -CCTG AvATCTGAGG Ara~GC~. GTATTACTGT 360 açr~raTa ATGGAGCGTA TTGTAGTGGT GGTAGCTGCT A~,~G~,G GTTcr-acccc 420 ~GrJG~ -G G~C~,vv, Q CC-v~ ~-. T Q GGTGAGT ACTGAATTCT Av~ GG 480 G~~a~~Cr~~ CCCTr-AACTT GG~.. GGGG rarGr-A~GC~ GCT~ r~TGA GGAa~GTGaC 540 Gcr~A~a~GT Ccarar,crAA TGCCQTvAG ccr~--AA~AT Gr-l~CCCTGAA C~ ,~ A~ 600 AGTTAAraA- Ct~aAGGGc~ GCI;~vG Gccra-GcTcT GTCC~ ararC GCGGTQQT 660 GrJrarraAC7T ~ v ;ACC cTcra~raa- GG---CrAq'CGG ~ 'C'CC~;. G~ra'CCTCC 720 T~r,raa~~--~a C~ vvGGG ra~a~CGCCC ;~vGG~-vCC TGGTQLGGA CTA ;--CCCC 780 G ~ vA Cvv-v~,v~v GAACTQGGC GCC~-~vACCA GC~G~;A QC~,.. ~CG 840 ~ v~ AC Av~ ;AGG ACTCTACTCC CTrar-~~~C'G TGGTGACCGT GCC;~,-.C~AGC 900 AG~.. ~;A cC-ra~~~c~a CATCTCCAAC CTCAATQQ a~Ccra~~aa ra~rar~¢TG 960 c-A~a-~ ~aa-- -.. v.vAGAG GCrar---a~a~- G----A~Cr'~ G ~v- v~,- vG aa~~ra~GCTC 1020 Avw~;~.v CX TG---A~C~C--a - ~CC~-~ A'r C~~Acccra~ Tcrar,G,--~raA- ra~ r~~~- 1080 CC~v.~,~vCC TCTTQCCCG GAGC~;. vC' C;~-CC~ T QTCCTQGG C-A~''~Gr,TCT 1140 TCTGGCl~ T~r,r~ra~GCTC Tr,-,~Aa~Gra ra~Gc~Tar~GT Cc~cc~ Aar~r rP~cc~c~,vc 1200 a~a~aaa~GC GCaG~.v~.v GGCTQGACC Tr~---Ca-Arl'A~C' QTATCCGGC a~~---A',CCTGC 1260 CccTr-arcTA r--C~--r~AC7CC AaArCcr,AAA ~ c~;Ar-Tc CCTCAGCTCv GACACCTTCT 1320 I;~C~CC ;AG ATTCQGTAA CTCCQATCT ~ ;A aAGccraAA~ v~vAQA 1380 AACTQQCA TGC-rrarrGT GCC~rar,GTAA Gcr-ar~ccr-AA~ GC ;-CGCC~;- CQGCTQAG 1440 GOOGrA~---a---a2- ,C:CC -.r~~~ TAG~;;.v~;AT cra~=--r ~ar Gcccr7~r~A-cG GGTGCTCAQ 1500 C~TC Q CCTC Q ~v~ 'C Tr-ar-rACCTG AA~ -~;~-GGG r~---r-aCCGTQ ~v~C~ - 1560 ~CC'CC~'-AAA ACC~AAr~G~-r~ ACCCT QTGA ~ 'c'~r~ hC CCCTGAGGTC A Q'~v~-v~vG 1620 W O 97/00079 PCTrUS96/10043 ~ G~ACGT ~r~rar~a GACCCTGAGG TCAAGTTCAA CTGvTACGTG r-a~GO~GG 1680 AVVTC~ATaA ~C~P--~~A AA~GGG ~--~--~~r,~A ~Ar~rl~G TA~'C~X~,~G 1740 TCAGC~-C~- CACC~.C~.C ~aCr~ ~T GGCTGAATGG r~ ,TAC AAGTC~a-~ 1800 TCTCr~ r~C~C~A GCC'CC~CG ~ -r~T CTCr~~C~ AAAC~v~ 1860 aGG~ cc~ r~ CATGr~-ara G~C~lOG cccr~-ccTc ,~CC~.~AGA 1920 GTG~~CG~TG Tp~~r-ApccTc ~V~c~Ar-~G GGr~CCCCG AnAAr,rarAr GTGTAr~~CC 1980 ,GCCCC~A~C CCC~_~TGAG cT~raAr-A ACCAGGT Q G CCTGACCTGC ~.vv.~fAAG 2040 G~-~ ATCC r~~Cr~~~TC vC~AGT CC~ T~G~c-A~CCr~ G~ ~aa~T 2100 ~-~-A-~r~G G~-'~v~G CTGGACTCCG AOGG~ .. ~..O~- --AC P~A~_CTCA 2160 A rar~rAr~GTGG r~ rJcGrA AC~-~ ~~C A~ C~-G ATGCATGAGG 2220 ~,~.A~-A~ CrarTarA~C r-A~Ar-A~C~ ~ C ~coGG~AaA TGA~TGCGAC 2280 a~ccc~c 2287 (2) INFORMATION FOR SEQ ID NOs9s ~i) SEQUENCF CHARACTERISTICS:
lAj L~NGTHs 442 amino acid~
~B~l TYPEs amino acid ~C, ST~ -CSs not r~.dr.L
~DJ TOPOLOGY: linear (ii) M~n~CnT-~ TYPEs prot~in (xi) 8EQUEN OE ~~DTPTIONs 8EQ ID NOs9s Ly~ Leu Thr Thr Met Asp Trp Thr Trp Arg Phe Leu Phe Phe Val Val Ala Ala Ala Thr Gly Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala &lu Val Lyn Lys Pro Gly Ser Ser Val Ly- Val 8er Cy~ Ly- Ala 8er Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly LQU Glu Trp ~Qt Gly Gly I1~ I1Q Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ala Arg Asp Ann Gly Ala Tyr Cyo 8er Gly Gly Ser - 42 ~
Cyu Tyr 8Qr Gly Trp PhQ Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val 8er 80r Ala 8er Thr Ly-~ Gly Pro 8er Val Phe Pro LQU Ala Pro 8er Ser Lys Ser Thr Ser Gly 61y Thr Ala Ala Leu Gly Cyl~ Leu Val ~ys Asp Tyr Phe Pro Glu Pro Val Thr Val 8er Trp A~n Ser Gly Ala ~eu Thr 8er Gly Val Hi~ Thr Phe Pro Ala Val Leu Gln 8er 8er Gly L~u Tyr 8er Leu 8er Ser Val Val Thr Val Pro Ser 8er 8er ABP Ly~

Lys Val Glu Pro Lys SQr Cys Asp Lys Thr Hi~ Thr Cy~ Pro Pro Cy~

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe LQU Phe Pro Pro ~y~ Pro Ly~ Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys ~al Val Val A~p Val 8er Hi-~ Glu ABP Pro Glu Val Lyl~ Phe A~n Trp Tyr Val A~p Gly Val Glu Val Hi~ A~n Ala Ly~ Thr Ly~ Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val LQU

Hi~ Gln Aep Trp Leu A~n Gly Ly~ Glu Tyr Lys Cy~ Ly~ VA 1 8~r Affn ~y~ Ala Leu Pro Ala Pro IlQ Glu Lys Thr Ile Ser Ly~ Ala Lyff Gly ~ln Pro Arg Glu Pro Gln Val Tyr Thr L~u Pro Pro SQr Arg A~P G1U

LQU Thr Ly~ A~n Gln Val Ser Leu Thr Cy~ Leu Val Ly~ Gly Pho Tyr Pro Ser A~p Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn A~n Tyr Lyll Thr Thr Pro Pro Val LQU A~p Ser Asp Gly Ser Phe Phe 385 390 395 ~00 ~eu Tyr Ser Ly~ Leu Thr Val A~p Ly~ Ser Arg Trp Gln Gln Gly A~n ~al Phe Ser Cys Ser Val Met HLs Glu Ala Leu Hi~ Asn HL~ Tyr Thr Gln Lyl~ 8er LQU Ser Leu Ser Pro Gly Ly W O 97/00079 PCT~US96/10043 (2) ~ ~TION FOR 8BQ ID NOslOs (i) SEQUBN OE rUARArT~TSTICSs ~A'l LENGTHs 1894 ba~e pair~
BI TY~Es n~ls~c ~cid C I STRP~ css singlQ
~DJ TOPOLOGYs linear ( ii ) ~ICIT.~T ~r TYPl!: S DNA ( j i C~ ) (xi) SEQU~N OE n~-~r~TPTION: SEQ ID NOslOs A.GvCv~- v~ C~.vvv.. ~-. TACAGTCCTG AvC~. ~.AC ~.~.v~.G~A PGCCr~~~TC 60 C Q .,v.v~G ~~CT~~T ACCvv.vCcC ATr-~Cr~A~G CC~f~C-~ 7A CCAGAT Q CT 120 vGCAAGTGGT TTTATATCGC A-.CGo~ ~r-AAA~rAr7G AGTArAATAA v.C4v..~AG 180 CAGATCCAAG Q AC~ .. TTACTTCACC ccrAAr~ rr~ r GA. ... C 240 r~~~~~TACC r~~~cr-~~A Gr-Acr-~-TGc ATCTA~AArA Cr~~CTP~cT GAATGTCCAG 300 C~C~~'TG Gr-~rJ~TCTC ~T~rGT~v G~-~GGCr~- AGCA...C~vC~ TCA~..v~.v 360 ATCCTCAGGG p~~~r~ CTACATGCTT G~-~ GACv Tr-AArr~TGA ~ ~T¢~v 420 CG6~v~ 7 TCTATC~TvA r~~Cr~~~~ ~r~~r~C P~CTGGv AGAGTTCTAC 480 GAAC~---~a A~v~-~vCYv QTTCCQAG TCAGATGTCG TGTAr~ TTGr~ ~ 540 ~'~T~r--TVT~7 ~r~~TGVA ~r--~r~ r--~ G~r'~~~r~G~ G~~CGGr~ 600 TCGGATCCCG AGGGTGAGTA CTAAr~CTTQ GCG~.~.GC CTGr-Ar~~AT CC~G~A~G 660 r~C~r~nT ~r~ AAr~ r-acc CCv- vCCT cTTr-Arc~GG AGC~ vCC 720 CGC~Cr~rTC ATGCT Q GGG ~ ~h.~ Gv~ CCr~~GCTCT G~C~~CC'~ 780 ~'CCT7~'GTG CCC~TAArCC ~_GCC~;A r~ AAr,GGG Qv~ aG GCTr~ CT 840 GCr~P~~CC ATA.C~GGvA Gr-Ar,CCTGCC CCTC~~CTAA GCCr~rCCCA APGGCC~ 900 TCTCCACTCC CTCAGCTCGv ACAC~ C~;~ 'C-;AGA TTCCAGTAAC TCCCAATCTT 960 ~, GCA~ P~CC~AAATC TT~vTC~AA ACTr~r~r~T G~CrArCGTG CCCAGGTAAG 1020 C~CCr-~-G C~-,~CC~,' QGCTQAGG CGGr-~r~r-GT GCC~T~r-~r-T AGC~ATC 1080 r~~Gr~~~~~, CCCCAr-CCGG ~.~.~A Q C GTC Q CCTCC A~ ~-, r~rr~rCTGA 1140 A~.C~.GGGG GGACCGT QG ~..C~. .. CCCCC'~AAA ccrAAr,rA~A CCCT Q TGAT 1200 ~.CCCG'-~~C CCTGAGGTCA Q-.GC~.~. GGTGGACGTG AGCCAr~-AAC ACCCTGAGGT 1260 CAAGTT QAC TGGTACGTGG A~GGC~.GCA GCTGrATAAT GCrAAr-ArAA ~GccGc~cr-~ 1320 G~PGC~C,TAC AArAr,rArGT ACC~G~.~. CAGC~-~.C ACC~.C~.GC ACr-~Gr-Ar,TG 1380 W O 97/00079 PCT~US96/10043 GCTGAATGGC aAr-r-~r-TACA AGTGCAAGGT CTC~Ar~AA G~C~.CC~AG CCCC~CGA 1440 ~ --r~C TCr~''-CC~ AAG~-~G~AC CO~.~4G~.G Cr-~rAGGC~r ATGr-~r~r-~r- 1500 GC~C~h-,OCG C~C'~CCTCT GCC~.~AGAG TC~-OCCTGT ~ -CTCT GTCCT~ 1560 G~r-'Y'CO~ r'-'-c TGT~-'-CCT GK'CC'C~CC CGGGATGAGC T-'-~''-~' 1620 CCAGGTCAGC CTGACCTGCC TGGTCAAAGG CTTCTATCCC ~-C~'-A~C~ CC~.~AGTG 1680 a--~--~----~T occ--~--ccc~ Ar~ArTA r~Ar~r~ ~--C~--GC TGGACTCOGA 1740 C~GL.C~.-C ..C~.~ACA G~CTCAC CGTGr-~r~Ar- AGCAGGTGGC ~--'-GrJr~' 1800 . ~A ~ 3C~-~A TGCATGAGGC TCTC-~-''- CAC~GC ~r~ ~c~ 1860 ~-.C~.~.~-- CC~.AAAT GAGTGCr-~rG GCCG 1894 (2) lh~ ~ATIoN FOR SEQ ID NOslls ~i) 8EQUEN OE ~U~T~TCTICSs ~AJ LENGTHs 437 amino ~cLd~
rB TYPEs ~mino ~cid ,C~ S~Nn~nNESSs not r~l~_~L
~DJ TOPOLOGYs linear ( Li ) MOT-~C~T-~ TYPBs prot~in (xi) SEQUBNCL D~CrPTPTIONs 8EQ ID NOslls Met Ala LeU 8er Trp Val Leu Thr Val L~U 8~r LOU L~U Pro LeU L U

Glu Ala Gln Ile Pro Leu Cy# Ala Asn Leu Val Pro Val Pro Ile Thr A~n Ala Thr Leu Asp Cln I 1Q Thr Gly Ly~ Trp PhQ Tyr Il- Ala 8er ~5 Ala Phe Arg A0n Glu Clu Tyr A~n Lys Ser Val Gln Glu Ile Gln Ala Thr Phe Phe Tyr Phe Thr Pro Asn Lys Thr Glu Asp Thr Ile Phe Leu Arg Clu Tyr Gln Thr Arg Gln Asp Gln Cy~ Il~ Tyr Asn Thr Thr Tyr Leu Asn Val Gln Arg Glu A~n Gly Thr Ile Ser Arg Tyr Vnl Gly Gly Gln Glu HL~ Phe Ala Hi~ Leu Leu Ile Leu Arg Asp Thr Ly~ Thr Tyr MQt L~U Ala Phe Asp Val Asn Asp Glu Ly~ A~n Trp Gly Leu 8er Val Tyr Ala Asp Lys Pro Glu Thr Thr Lys Glu Gln LQU Gly Glu Phe Tyr Glu Ala Leu Asp Cys Leu Arg Ile Pro Lys Ser Asp Val Val Tyr Thr Asp Trp Lys Lys Asp Lys Cys Glu Pro LQU Glu Lys Gln Hi~ Glu Ly~

alu Arg Ly~ Gln Glu Glu Gly Glu 8~r A~p Pro Glu Gly Glu Pro Ly~

S~r Cys Asp Lys Thr E~is Thr CYB Pro Pro Cy8 Pro Aln Pro Glu Leu LQu Gly ¢ly Pro 8Rr Val Ph~ IA~U PhQ Pro Pro Lys Pro I.y~ Asp Thr ~Qu M~t I 1~ 8er Arg Thr Pro Glu Val Thr Cy~ Val Val ~al Asp Val ~~r Hi- Glu Asp Pro Glu Val Lys Ph~ Asn Trp Tyr Val Asp Gly Val Clu Val EIis Asn Ala Lys Thr Lys Pro Arg Glu Glu ¢ln Tyr A~n Sor Thr Tyr Arg Val Val S~r Val L~u Thr Val Lou His Gln Asp Trp L~u Asn Gly Lyi~ Glu Tyr Lys Cy8 Lys Val S~r Asn Lys Ala L~u Pro Ala ~ro Ilo Glu Ly~ Thr Ilo 8er Lyt~ Ala Lys Gly Gln Pro Arg Glu Pro ~ln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Ly~ A~n Gln Val SQr LQU Thr Cy~ Lau Val Lya Gly ~PhQ Tyr Pro S~r A~p I 1Q Ala Val Glu Trp Glu S--r Asn Gly Gln Pro Glu Asn A-~n Tyr Lyff Thr Thr Pro Pro Val L~u Asp Ser Asp Gly S~r Ph~ Ph~ L~u Tyr Slar Lys L~u ~hr Val A~p Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 8~r Cy~ S~r ~al N~t His Glu Ala L~u HLs Asn EIi8 Tyr Thr Gln Lys 8~r ~au Scr L~u S~r Pro Gly Lys (2) lN~.~ATION FOR SEQ ID NOsl2s i ) SEQUENOE CHARACTkRISTICS s lA' LENGTH: 442 amino acids B, TYPE : amino acid ~C, sT~ n~ lzcSs not rolHvant ~D~ TOPOLOGYs lin~ar TYPE s prot~in CA 0222462~ 1997-12-12 W O 97/00079 PCT~US96110043 (xi) 8EQU~N OE ~-C~TPTIONs SEQ ID NOsl2s Ly~ L u Thr Thr ~et Aop Trp Thr Trp Arg Phe Leu Phe Ph~ Val Val ~la Ala Ala Thr Gly Vnl Gln S~r Gln Val Gln Leu Val Gln 8Qr Gly Aln Glu Val Ly~ Lys Pro Gly Ser S~r Val Lys Val Ser Cy8 Lys Ala Ser Gly Gly Thr Phe 8er Ser Tyr Ala Il- Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Lou Glu Trp ~et Cly Gly Ilo Ile Pro Ile Pho Gly ~hr Ala Asn Tyr Aln Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala ~~p Glu Ser Thr Ala Arg Asp Asn Gly Ala Tyr Cy~ Ser Gly Gly Ser Cys Tyr 8Or Gly Trp PhQ Asp Pro Trp Gly Gln Gly Thr Lou Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Ly~ Ser Thr Ser Gly Gly Thr Ala Ala Lou Gly Cyo L~u Val ~yo Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn SQr Gly Ala ~~u Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Sor Val Val Thr Val Pro Ser Ser S~r A~p Lyu Ly- Val Glu Pro Lys S~r Cy8 Asp Lys Thr Uis Thr Cy~ Pro Pro Cy-Pro Ala Pro Glu Leu L~u Gly Gly Pro Ser Val Phe Leu Phe Pro Pro ~y~ Pro Ly- A~p Thr Lou Nbt Ile Sor Arg Thr Pro Clu Val Thr Cy~

~al Val Val Asp Val Ser His Glu Asp Pro Glu Val Asn Phs Ser Trp Tyr Val Asp Gly Val Glu Val His Asn Asn Lys Thr Lys Pro Arg Glu Glu A~n Tyr Ssr Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Hio Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lya Cys Asn Val Ser Asn W O 97/00079 PCT~US96/10043 Ly0 Ala LQu Pro Ala Pro Il~ Glu Lys Asn Il~ 8~r ~y~ Ala Lys Gly ln Pro Arg alu Pro aln Val Tyr Thr L~u Pro Pro S~r Arg Asp alu Lou Thr Lys Asn Gln Val 8~r Leu Thr Cy~ LQu Val Ly~ Gly Ph~ Tyr Pro S~r A~p Il~ Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu A~n A~n Tyr Ly~ Thr Thr Pro Pro V~l L u Asp 8~r Asp ¢ly 8Or Pho Pho Lou Tyr 8er Lys Leu Thr V 1 Asp Ly~ Ser Arg Trp Gln Gln Gly A~n Val Phe Scr Cys sQr Val ~t ~is Glu Ala Leu His Asn His Tyr Thr , 420 425 430 Gln Lys 8er Lou Ser Leu Ser Pro Gly Lys 435 ~40 (2) INFORMATION FOR SEQ ID NO t 13s (i) SEQUEN OE rUA~CT~RTCTICSs ~A' LENGTHs 42 amino acid~
IBI TYPBs ino acid ,CJ ST~ ASS not ~al~.~n~
~DJ TOPOLOGYs linQar ~ii) M~T~C~T-~ TYPEs prot~in (xi) SEQUBN OE ~C~RTPTIONs SEQ ID NOsl3s Pro alu Mct L u Arg Asn S~r Thr Asp Thr Thr Pro L~u ~hr aly Pro Gly Thr Pro Glu S~r Thr Thr Val Glu Pro Ala Ala Arg Arg sQr Thr aly L~u A~p Ala Cly Gly Ala Val Thr Glu ~2) l~ndATION FOR SEQ ID NOs14s (i) SEQUEN OE C~ARACT~RISTICSs ~AJ LENGTHs 16 am~no acids IBI TYPEs amino acid ,C, S~R~Nn~n'~CSs not rQlQvant ~DJ TOPOLOGYs linoar (ii) ~nT.~.~ TYPEs prot~in (~i) SEQUEN OE D~-C~-RTPTIONs SEQ ID NOsl4s LQu Thr Thr Glu L~u Ala A~n NQt Gly Asn L~u S~r Thr Asp S~r Ala

Claims

- 48 -

1. An organic molecule to which there is covalently bonded a sialyl-Lex determinant and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on said molecule.

2. The organic molecule of claim 1, wherein said molecule contains multiple sialyl-Lex determinants or multiple sulfated determinants.

3. The organic molecule of claim 1, wherein said molecule is soluble.

4. The organic molecule of claim 1, wherein said molecule comprises a P-selectin ligand consisting essentially of amino acid 21-57 of Fig. 8A, or a portion thereof which is capable of mediating an interaction with the P selectin receptor.

5. The organic molecule of claim 4, wherein said molecule comprises a P-selectin ligand consisting essentially of amino acids 38-57 of Fig. 8A.

6. The organic molecule of claims 1 or 4, wherein said molecule comprises .alpha.1-acid glycoprotein (AGP).

7. The organic molecule of claims 1 or 4, wherein said molecule comprises an antibody molecule.

8. A purified nucleic acid encoding any of the organic molecule of claims 4-7.

9. The purified nucleic acid of claim 8, wherein said nucleic acid further encodes either (a) .alpha.1-acid glycoprotein (AGP) or (b) an antibody molecule.

10. A vector comprising the nucleic acid of claim 8.

11. A cell comprising the purified nucleic acid of claim 8.

12. A method of inhibiting the binding of a cell bearing a P-selectin protein to a molecule or cell bearing a sialyl-Lex determinant and a sulfated determinant, said method comprising contacting said P-selectin protein-bearing cell with an organic molecule of claim 1.

13. The method of claim 12, wherein said organic molecule also inhibits the binding of a cell bearing an E-selectin protein to a molecule or cell bearing a sialyl-Lex determinant.

14. A method of reducing inflammation in a mammal comprising administering to said mammal a therapeutically-effective amount of an organic molecule of claim 1.

15. A method for reducing or protecting a mammal against an extravasation-dependent adverse reaction, said method comprising administering to said mammal a therapeutically-effective amount of an organic molecule of claim 1.

16. The method of claim 15, wherein said extravasation-dependent adverse reaction is extravasation-dependent organ damage or clotting associated with adult respiratory distress syndrome, glomerular nephritis, or ischemic myocardial injury.

17. A method for reducing or protecting a mammal against an adverse immune reaction, said method comprising administering to said mammal a therapeutically-effective amount of an organic molecule of claim 1.

18. The method of claim 17, wherein said adverse immune reaction is induced a microbial factor or a host factor.

19. The method of claim 17, wherein said adverse immune reaction is septic shock or septicemia.