CA2202472A1 - Hematopoietic cell l-selectin ligand (hll) and therapeutics thereof - Google Patents

Abstract

An isolated and purified glycoprotein and functional analogs are disclosed. The glycoproteins are characterized by being expressed on at least primitive hematopoietic cells, and being a ligand for L-selectin. The binding of ligand to L-selectin is not inhibitied by anti-CD34 antibodies nor by MECA 79 monoclonal antibody.

Description

1.

HEkl~TOPOIE:TIC CE:LI- L-8E:L~ N I.IGAND
~liI-) AND T~E:RAPEtrq!IC8 THBRE:OF

R~R~ROC12nD OF l~le l~V~'ON

TECHNICAL FIELD

The present invention involves the development of compounds which can regulate and control the function of adhesion molecules.

BA~K~-~OUND ART

The adhesion molecules are involved in the fundamental control of cell-cell interaction and cellular migration. Adhesion molecules regulate diverse proc~s~e- in inflammation, hematopoiesis and tumor metasta6is. (Woodruff, et al, 1987; Springer, et al, 1987; Sharon and Li~, 1993; Scakstein, 1993) For general review6 on adhesion molecules see Carlos and Harlan, 1994 and Chin et al, 1991. It would be u6eful to develop reagents which can control and regulate the adhesion proteins, particularly within the selectin family.

suBsrllurE SHEET (RULE 26 .

, CA 02202472 1997-04-11 WO96/11012 PCT~S95/13736 Tle peripheral lymph node "homing receptor", L-selectin (CD62L), is a -75 kDa glycoprotein which mediates attachment of lymphocytes to lymph node (LN) high endothelial venules (HEV), an adhesive interaction which is the fir6t step in the migration of lymphocytes from blood into lymphoid ti6sues (Gowans and Knight, 1964; Marchesi and Gowans, 1964). This trafficking of lymphocytes from blood into lymph nodes is markedly nonrandom and is initiated by specific adherence of the lymphocytes to HEV. The "lymph node homing receptor" or L-6electin (LECAM-l) i6 the principal lymphocyte membrane glycoprotein mediating this attachment.
The L-selectin protein is recognized by a variety of monoclonal antibodies (mAbs) in humans {Gatenby et al., 1982 (Leu-8); Reinherz et al., 1982 (TQ-l); Tedder et al., 1990 (LAM)} and is a member of the selectin family of adhesion molecules, which includes P-selectin (CD62P) and E-6electin (CD62E). Selectin6 6hare a common structure consisting of an amino-terminal calcium-dep~nAPnt lectin domain, an epidermal growth factor domain, a variable number of repeat sequences bearing homology to complement regulatory and catalytic proteins b~ ~A~n~ C3b or C4b, a transmembrane portion, and a C-terminal cytoplasmic SUBSrlTUTE SHEET (RUL~ 26~

Wos6/llol2 PCT~S95/13736 tail (Bevillcqua and Nelson, 1993; Rosen, 1993).
The molec~llA~ weight varies among leukocytes due to posttranslational glycosylation among ~ubsets of leukocytes.(Carlos and Harlan, 1994) The lectin domain of these proteins directs their adhesion to carbohydrate molecules pre6ent on the cell surface.
The adhesive interaction between lymphocytes and HEV has been extensively analyzed u~ing an in vitro bi~in~ a~6ay (Stamper and Woodruff, 1976). This a6say is performed under shear at 4C, whereby binding mediated by L-selectin i~ maximized and effects of other adhesion molecules are minimized (Shaw et al, 1986; Spertini et al., 1991). The interaction of L-selectin with its corresponAi n~ ligand(s) on HEV i~ calcium-dep~n~nt (Woodruff et al., 1977) and requires the presence of sialic acid (Rosen et al., 1985; True et al., 1990) and ~ulfate (Imai et al., 1993) on the ligand(s~. L-selectin behaves a~ a lectin and recognizes sialylated, high mannose residue~ on its corresponding ligand which i5 identified by the monoclonal antibody MECA-79 (Sack~tein, 1993).
MECA-79 identifie~ an L-~electin ligand on lymph node HEV and which cross-reacts with GLYCAM-l and CD34. In v~tro adherence of lymphocytes via L-selectin can be inhibited by carbohydrate6 such a6 mann~-~ 6-pho~phate (man-6-P), PPME (Phosphomannan SUBSnTUTE SHEET (RULE 26) W096/11012 PCT~S9Sl13736 monoester core from Hansenula hostii, a phosphomannosyl-rich polysaccharide), and fucoidin (a sulfated, fucose-rich polysaccharide) (Stoolman and Rosen, 1983).
Ligands for L-selectin have thus far been characterized on murine endothelial cells utilizing a murine L-selectin IgG chimera molecule as a probe. (Watson et al., 1990) This approach has identified two proteins, GlyCAM-l (SgpS0) (Imai et al., 1991) and CD34 (Sgp90) (Baumhueter et al., 1993), present on endothelial cell6. GlyCAM-1 is a novel sialomucin, and its role as a ligand for L-selectin is its only known function (Lasky et al., 1992). Although present on endothelial cells in most tissues (Beschorner et al., 1985), CD34 is best known for its expression on the earliest multilineage colony-forming hematopoietic stem cells (Civin et al., 1984).
Hematopoietic progenitor cell~
characteristically express both L-selectin and CD34 (Terstappen et al., 1992), and there is growing evidence that L-selectin plays a role in hematopoiesis (Terstappen et ~l., 1993; Kobayashi et al., 1994). The characterization of L-6electin and its ligands among progenitor cells is of considerable interest as adhesion proteins regulate Sl~TUTE SHEE~ (RULE 26) ~ CA 02202472 1997-04-11 Wo96/11012 PCT~S95/13736 cell-cell and cell-stromal interactions fundamental to hematopoiesi~.
In general assays for determining the adhesion between lymphocyte and HEV requires the use of frozen-gections of lymph nodes.(Stamper and Woodruff, 1976; Sackstein et al, 1988) It would be useful to be a~le to use cells in suspension in the a~say also. This would enable the use of cell lines, giving ri~e to more reproducible result~ as well as reducing the need for surgical procedures for lymph node removal.
It would be useful to have ~trategies which would allow regulation of hematopoiesis cince it i6 regulated by cell-cell and cell-stromal interaction~. For example, Terstappen et al (1993) have shown that activation of L-selectin increases the clonogenic capacitiy of stem cells.
During recovery of immune function following bone marrow transplantation pathologic changes have been observed following transplantation which interfer with lymphocyte migration and HEV integrity. Further, in addition to changes in lymph node structure, alterations in lymphocyte migration can occur ~econAAry to the effect of pharmacologic agent6 used in posttransplant therapy such as corticosteroid6 (Sackstein, 1993). It would be useful to ha~e an SUBSTITUTE SHEET (RULE 26) WO96/11012 PcT~S9Sl13736 agent which can assist in reestablishing lymphocyte trafficking and 80 immune function following bone marrow tran~plantation.
The crucial role of adhesion molecules in controlling and directing the inflammatory process indicate~ that a reagent which interfere~ with the proce~s, i.e. anti-adhesive, could have anti-inflammatory properties.
Further, cell adhesion molecules are involved in metastasis, therefore it would be u~eful to develop an anti-adhesive which has anti-metastatic properties. In particular, with the identification of L-selectin on hematopoietic cells, it would be useful to have an anti-adhesive that affects L-~electin in leukemia to decrea~e the growth and ~pread of malignant hematopoietic cell~
throughout the body.
Further, it would be useful to have additional cell marker~; and monoclonal an~; hoA; es directed against these cell markers to allow for cell targeting.

SUBSrlTIJTE S~IEEt (RULE 26) Wo96/11012 PCT~S95/13736 .

8~MMARY OF THE lNv~..~ON AND ADvaNTAGE8 According to the present invention, an isolated and purified glycoprotein and functional analogs are disclosed. The glycoproteins are characterized by being expressed on at least primative hematopoietic cell6, and being a ligand for L-selectin. The binA;n~ of ligand to L-selectin i~ not inhibited by anti-CD34 antibodies nor by MECA-79 monoclonal antibody.

BRIEF D -~PTPTION OF T~E DRA~ING8 Other advantages of the pre~ent invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

FIGURE lA-B are photomi~G~L~ph6 of cytospin preparations of RGla cells demonstrating adherence of lymphocytes (6mall dark dots), (A) Lymphocytes adhere to KGla in the pre6ence of CD45 or i60type control Abs, (B) Lymphocyte binding a6say in the presence of LAN1-3 Ab (anti-L-selectin);

SUBSTITUTE SHEET (RULE 261 , CA 02202472 1997-04-11 W096tl1012 PCT~S95/13736 F~GURE 2 is FACS profiles of lymphocytes used in the binding assay after incubation with isotope-matched IgG control, LAM1-3, or anti-CD45 Abs, followed by GAM-FITC, results shown are representative of 3 independent experiments;
FIGURE 3A-D are FACS profile6 of KGla cells sorted by FACS prior to the binding assay into CD34+ and.CD34- fractions using mAb HPCA-2PE, sorted cell fractions were restained for CD34 using mAb QBENDlO-FITC and analyzed, positive and negative sorted fractions were >90% and <lO~
positive for CD34, respectively, results shown are representative of 3 independent experiments;
FIGURE 4 i8 a photomicrograph showing the lymphocyte adherence assay performed on the sorted cells, and no differences in lymphocyte adherence were evident among the CD34+ and CD34- populations, adherence to the CD34 negative fraction is shown;
and FIGURE 5A-F are FACS profiles of COS-7 cell~ were transfected with either CD34-pCDM8 (E,F) or pCDM8 (mock~ C,D), then analyzed by FACS and compared to KGla (A,B) for CD34 expression, Abs used were isotype-matched IgGl ~u.lLLol and anti-CD34 mAb QBENDlO, Lymphocytes did not adhere to CD34-transfected COS-7 cells, despite higher levels of CD34 expres6ion a6 compared to KGla cells.

SUBSTITUTE SHEET (RULE 26~

D~T~TT~n DE8CRIPTION OF THB PRE:FERRED E:NBODIME:NT

The present invention provides an isolated and purified glycoprotein and functional analogs thereof. Analog is defined as a molecule that will be generally at lea6t 70~ homologous over any portion that i5 functionally relevant. In more preferred embodiments the homology will be at least 80% and can approach 95% homology to the amino acid sequence of the protein segment of the glycoprotçin. The homology will extend over a region of at least 8 contiguous amino acids to 80 contiguous amino acid~. The amino acid 6equence of an analog may differ from that of the glycoprotein of the present in~ention when at least one residue is deleted, inserted or substituted. The molec~lAr weight of the glycoprotein may vary between the analog and the present invention due to carbohydrate differences. Differences in glycosylation may be present between the analog and the present invention.
The gly~oproLein has the folIowing functional characteri6tic6. It is expres6ed on at least primative hematopoietic cell6. The gly~o~o~ein is a ligand for L-6electin. The ligand binding to L-select1n i8 not inhibited by anti-CD34 ant~hoAies and is not reco~n~zed by the SUBSrlTUTE SHEET ~RULE 26) Wo96/11012 PCT~Sg5/13736 . I
MECA-79 monoclonal antibody. The glycoprotein is designated hereinafter as hematopoietic cell L-selectin ligand, HLL.
Further, the glycoprotein, HLL, is a membrane associated glycoprotein and functions as an adhecion protein ligand. The gly~oplGLein facilitates attachment of lymphocyte~ to hematopoietic cells including primitive hematopoietic cells.
The present invention also provides for an antibody directed against the glycoprotein, HLL.
The anti hoA~ es may be either monoclonal or polyclonal. Murine monoclonal ant~boA~es are initially r~; fi~A against RGla cells. The monoclonals that are generated are then screened for the ability to- block lymphocyte binding to KGla.
Utilizing the~e monoclonal antiho~est the gl~o~G~ein is isolated by immunoprecipitation of KGla membrane lysates as is stA~A~rd in the art and used for the production of further antibodies as needed. Such methods can be found described Sambrook et al, Molecular Clon~n~: A L&boratory MPn~7 ~ Cold Springs Harbor, New York, 1989, as well as additional methods of isolation and purification as are known in the art.

SUBSTITUTE SHEET (RULE 26) W096/11012 PCT~S9S/13736 Additionally, the antibodies may be prepared against a synthetic peptide based on the 6equence, or prepared recombinantly by cloning ~er-h~ ques or the natural gene product and/or s portions thereof may be isolated and used as the immunogen. Such proteins or peptides can be used to produce antibodies by stAn~rd antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Ant~ho~;es: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988.
For producing polyclonal antihoAies a host, such as a rabbit or goat, is immunized with the protein or peptide, generally with an adjuvant and, if neces~ary, coupled to a carrier; ant;ho~;es to the protein are collected from the sera.
For producing monoclonal antibodies the te~n;gue involves hyperimmunization of an a~plo~iate donor with the protein or peptide fragment, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody.
The cells are then cultured, in bulk, and the monoclonal ant1hoA~e6 harvested from the culture media for use.

sussnTuTE SHE~ (RU~E 26) WO96/11012 PCT~Sg5/13736 The antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated a6 is well known in the art. (For a general discu~sion of conjugation of fluorescent or enzymatic moieties see Johnstone & Thorpe, Immunochemistry ~n Pr~ctice, Blackwell Scientiflc Publlcations, Oxford, 1982.) The binding of an~ihoA~efi to a ~olid DU~O~ ~ substrate i6 also well known in the art. (see for a general ~irc~ ion Harlow & Lane Ant;ho~ies: A L~boratory Mqm~AI, Cold Spring Harbor Laboratory Publications, New York, 1988) The detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline pho~phata~e, B-galactosida~e, peroxida6e, urease, fluore6cein, rhodamine, tritium, 14C and iodination.
The method of targeting cells includes the step6 of preparing an~ho~e6 directed against the gly~o~L~e~n as described above and coupling the ant~h~ies to the a~op~iate agent whether for cell killing, cell selection or cell identification. For cell k~ to~Y~nR such a6 ricin A chain, ~ omonas exotoxin A, diphtheria toxin, other plant and bacterial toY~n~ a6 well as SUBSTIT~E S~IET (RULE 26) chemotheraplutic compounds can be coupled in the present invention forming an immunotoxin. For a general review of the antibody-toxin art see Ramakr~h~n, 1990.
Cell targeting requires exposing a population of cell~ to the immunotoxin. A toxin bound antibody can be administered to the appropriate patient and targeted cells killed ~n v~vo. The immunotoxin is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, ~r~ ng of administration, and other factors known to medical practitioners. The "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve at least 25% of the treated patient~ exhibit im~ovement including but not limited -to improved survival rate, more rapid recovery, or improvement or elimination of 6ymptoms.
Alternatively, cells can be removed from the patient and treated ex v~vo selectively. For example, cells expressing HLL can be removed through complement-mediated lysis from the ex v~vo population and the remaining cells r eL~L..ed to the patient. Additional cell removal can be undertaken SUBSTITUTESHEET~InE2~

W O96/11012 -14- PCT~US95/13736 utilizing ce~l sorting, l~pAnning~ and magnetic bead separation. Further, utilizing cell sorting, ~lp~nning~l magnetic bead separation and the like cell populations can be enriched for HLL bearing cells and this enriched cell population returned to the patient.
The targeted cells to be removed are cell~ expressing HLL and can be selected from the group consisting of leukemic cells, malignant hemopoietic progenitor cell~, or any malignant cell expressing the marker.
The present invention also provides a method of regulating hematopoiesis, particularly in reconstitution of the immune system following bone marrow transplantation. The present invention includes the steps of selecting those cells with high(+) or low(-) expres6ion of HLL depenAing on the growth characteristics associated with the marker density needed by the patient. The selection proGed~e utilizes ex v~vo methods a6 described herein. After selection, the selected cell type is cultured ~n v~tro, if ne~A to eYp~nA
the population using s~A~rd methods known in the art. The patient is then infused with the ~rAnAPA, enriched HIL+ or HLL- population as n~eA~d ~

SUBS~lTUTE SHEE~ (RULE 26) Wo96/11012 -15- PCT~S95/13736 T~e present invention further provides a method of regulating inflammatory re~ponse by interrupting cellular migration into lymph nodes and sites of both acute and chronic inflammation including the step of administering to the patient antibody directed agains HLL thereby disturbing cellular migration mediated by ~-T- by blocking the the lymphocyte attachment site and can be injected directly at the inflammed ~ite if nee~eA. The regulation of the inflammatory response would be useful in autoimmune disorders, post-ischemic tis6ue injury and sepsis (Calos and Harlan, 1994).
Administration and effective dose are as described for immunotoxins hereinabove.
Studies by applicant on the interaction of L-selectin and hematopoietic CD34 function as an adhesive receptor-ligand pair, in v~tro binding studies of lymphocytes to XGla, a primitive CD34-positive human cell line derived from an acute myeloid leukemia (Civin et ~1., 1994; Koeffler et al., 1980) lead to the pre~ent invention. These studies surprisingly revealed highly specific adherence of lymphocytes to KGla cells mediated by L-selectin on the lymphocyte, but uneYrectedly not involving CD34 as the corresronA~ ligand as had been previously Le~O~ Led (Baumhueter et al, 1993;
Oxley and Sackstein, 1994). The result6 indicated SII~ITUIE S~IE~T (Rl~E 26) W O96/11012 PCTnUSg5tl3736 j the presence of a ligand, designated HLL, for L-selectin on the surface of this hematopoietic progenitor cell line and provide the fir~t evidence of L-selectin-mediated adhesion between lymphocytes and a non-endothelial cell type.
For these 6tudies, the lymphocyte-HEV
adherence assay was used which is an in vitro approximation of physiologic adhe6ion mediated by L-~electin was used. It ha6 been a fundamental tool in 6tudying the function of L-6electin in its native state on the surface of lymphocytes. This binding assay was novelly adapted to examine lymphocyte-hematopoietic cell adhesion, and the results provide the unexpected results of L-selectin-dependent adhesive interactions between lymphocytes and non-endothelial cell6.
The adaptation of the assay allowed for the first time the u~e of cell lines in a lymphocyte-HEV adhe~e,.ce assay. In this assay, slides were prepared of KGla cell suspensions which were used in place of slides of frozen lymph node sections a8 taught by the prior art. The KGla cells were placed on the slides by using cyto-spin centrifugation as further described herP~nhPlow.
Several ~n~Pp~n~ent lines of evidence indicate that lymphocyte b~n~1ng to RGla is mediated primarily, if not 6clely, by L-~electin.

SUBSrlTUI~ SHEET (F~LILE 26) Wo96/11012 -17- PCT~S95/13736 Fir6t, an anti-L-6electin mAb (LAM1-3) previously shown to block L-selectin-mediated adherence to LN
HEV tspertini et al., 1991), completely inhibited PBL from binding to KGla or LN HEV, wherea6 anti-CD45 and i60type control Abs did not blocklymphocyte binding. Second, L-selectin-mediated binding is a calcium-dependent event, and lymphocytes were unable to bind to KGla in the presence of the calcium chelator EDTA. Third, carbohydrates 6uch as man-6-P, PPME, and fucoidin inhibited lymphocyte adherence to RGla. These compounds are all known to bind to L-6electin and to inhibit lymphocyte binding to HEV in the in vitro a6say (Stoolman and Rosen, 1983; Stoolman et al., 1984). Lastly, it is known that PMA treatment of lymphocytes causes shedding of membrane L-selectin via a protein kinase C activation pathway, and corre6ponds to the loss of lymphocyte binding to LN HEV in the ~n v~ tro as6ay (Tedder et al., 1990). In these studies, PNA-treated PBL were no longer able to bind to XGla.
The nature of the ligand wa6 investigated by determining the effect6 of various enzyme treatment6 of KGla on the binding capacity.
Previou6 6tudies have shown that ligand expres~ion of sialic acid is es6ential for L-selectin-mediated b~ n~ ~ ng of lymphocytes to LN HEV (Rosen et al., SUBSrlTU~ SHEET ~RlllE 26~

W O96111012 PCTrUS95/13736 1985). In the pre6ent example6, neuraminida6e-treated KGla showed a complete 1058 of lymphocyte binding, indicating that 6ialic acid re6idue6 are al60 a neces6ary component on the RGla L-6electin ligand; a6 ~uch, lymphocyte adherence to KGla involves carbohydrate motif6 and i6 not ba6ed 6trictly on protein-protein interactions. This fin~;ng, combined with the re6ults of protease experiment~, indicate6 that the KGla ligand i8 a 10 glycoI~GLein.
To examine whether 0-linked glyco6ylations on the ligand play a central role in the adhesive interaction, KGla were digested with the enzyme 0-fiialoglycu~LoLein endopeptidase which 6pecifically cleave6 protein6 at 6ite6 of o-linked sialo-glycosylation (Abdullah et al., 1992) and which has been shown to differentially cleave epitope6 of the CD34 molecule (Sutherland et al., 1992). The data reveal that treatment of KGla in suspension with the enzyme actively de~LLoyed CD34 epitopes, yet had no effect on lymphocyte adherence. The6e re6ult6 sugge6t that ligand sialic acid residues critical to b~t~Ai nq are pre6ent on N-linked rather than on 0-linked glycosylations.
CD34 has been Le~oLLed to be a ligand for L-selectin hAr~ on the f1n~ng that a murine L-SVBS~JTE SHEET (RU~E 26) Wo96/11012 PCT~S9~/13736 selectin-IgG chimera molecule precipitated CD34 from a murine lymph node lysate (Baumhueter et al., 1993). The result6 a~ set forth in the examples indicate that CD34 as expressed on KGla is not a functional ligand for lymphocyte L-selectin, as no difference in lymphocyte binding to sorted CD34-and CD34+ KGla cells was observed.(F-igures 3 and 4) Titration studies using varying proportions of KGla and HL60 have demonstrated that the amount of lymphocyte adherence is directly proportional to the percentage of input KGla cells, indicating that differences in lymphocyte binding to the positive and negative sorted fractions would have been evident if CD34 were the ligand. It is unlikely lS that a particular bi nAi nq epitope of CD34 as selected, as this experiment was done using two different anti-CD34 mAbs to sort the XGla. Two forms of CD34 on XGla have been reported (ntruncated" and "full lengthn) (Xrause et al., 1993); however, these differences do not account for the data here as sorting was also performed u~ing QBENDlO, which recoqn1~es both forms.
In addition to sorting experiments, evidence that CD34 i8 not the L-selectin ligand on KGla is derived from mAb bloçk~ng studies and adherence assays using other CD34 positive cell6.
None of the anti-CD34 mAh8 tested, or any SUBSl ITUTE SHE~T (RULE ~6) Wo96/11012 PCT~S95/13736 combination~thereof, was able to block lymphocyte binding to KGla. Furthermore, lymphocytes did not adhere to another primitive CD34+ cell line, RPNI
8402, and tran6fection of CD34 into COS-7 cells did not confer lymphocyte binding capacity. While potential glycosylation differences of the CD34 molecule expressed by the~e cell types could affect their ability to support lymphocyte adherence, thi~
explanation i6 ~ kely in light of equivalent adherence observed among the sorted CD34+ and CD34-KGla cell6. Taken together, the data presented here indicate that the CD34 glycoform present on hematopoietic cell6 is not a ligand for L-selectin.
Moreover, flow cytometric analy~is of the various lS cell lines utilized in the bi n~ i ng assay provides evidence that membrane structures such as LFA-l, VLA-4, CD44, Sialyl LeX and CD43 do not play a primary role in lymphocyte adherence to KGla since each of these molecules were also present on at least one other cell line te~ted that did not demonstrate lymphocyte b; nAi n~.
HLL is not recognized by MECA-79 monoclonal antibody which identifies L-6electin ligands on lymph node HEV. ImmunofluorP~cence analysi~ of KGla using NECA-79 shows no evi~nce of the protein identified by NECA-79. HLL is shown to SUB~E SHEET (~lJI E 26~

W O96/11012 P~-lr~S5~/13736 be unique from L-6electin ligands thus far identified.
In the present study, direct cell-cell interactions were utilized to detect the presence of an L-selectin ligand on a hematopoietic cell.
Other studies directed at identifying L-selectin ligands have relied on moler~lAr approaches utilizing a murine L-selectin-IgG chimera molecule, synthesized in a human embryonal kidney cell line, as a probe (Watson et al., 1990). Of note, studies utilizing this chimera have failed to demonstrate binding of the molecule to KGla cells (Majdic et al., 1994). In general, tissue- and species-specific patterns of glycosylations are well described, (Yamashita et al., 1983; Cullen et al., 1981; Yamashita et al., 1985) and such differences can affect the biological activity of proteins expressed in different cells (Cowing 1983; Huff et al., 1983). A6 it i6 known that glycosylation of L-selectin varies among different cell6 expressing the protein (Lewinsohn et al., 1987, Ord et al., 1990; Griffin et al., 1990), such differences may a~o~lL for the observation here that native L-~-lectin, expressed on lymphoc-;e membranes, selectively binds to a corr~pQnA~r~ ligand on KGla cell6 while the chimera apparently does not.
Similarly, differences in glycosylation of CD34 SU~STlTUTE SHEET (RULE 26) W096/11012 PCT~S95/13736 among endot1elial cells and hematopoietic cell6 may account for the differential capacity of this protein to participate in L-selectin interactions among these cell type~.
L-6electin ligands have been recognized heretofore only on endothelia-l cells. The detection of an L-selectin ligand on a non-enaothelial cell eYp~nA~ the physiologic implicatiQns of L-selectin function beyond it~
well-characterized role in regulating leukocyte trafficking.
The above discussion provides a factual basis for the characterization and use of }~-T-. The methodc used with and the utility of the present invention can be shown by the following examples.

~ raXPLE8 GENER~T METHODS:
Cell L~ne~. Cell lines u~ed in these studies were obtained from the following sources:
KGla and Nalm 16, gift of Dr. William E. Janssen;
HL60, K562, and Ra~i, gift of Dr. Lynn Moscin~ki;
COS-7, gift of Dr. Ke~th Zukerman (all from H.
Lee Moffitt CAncer Center, Tampa, FL); RPMI 8402, gift of Dr. Daniel G. Tenen ~Harvard Medical School~ Boston, MA). All cell6 were cultured in RPMI 1640 (Gibco-BRL, Gaither~burg, MD) SllBSnTU~E SHEET (RULE 26) Wo96/11012 PCT~Sgs/l3736 .
6upplemente with 10% heat-inactivated fetal bovine serum (FBS) in a humidified chamber at 37C with 5%

C2 in air.
Preparat~on of Lymphocytes. Human S peripheral blood lymphocytes (PBL) were i60lated by Ficoll density gradient from blood drawn in sodium citrate. To obtain rat thoracic duct lymphocytes (TDL), thoracic duct~ sf rats were cannulated as described by Bollman et al.(1948). Lymph was collected in phosphate buffered saline (PBS) with O.l~ penicillin/streptomycin and 5 U/ml heparin.
PBL or TDL were washed three times in RPMI 1640 medium without bicarbonate (Gibco-BRL), pH 7.4, and suspended at l x 107 cells/ml in above medium with s% FBS and kept on ice until u6e in the adherence assay.
Lymphocyte Adherence As6~y. The procedu~e for the ~n vitro binding of human or rat lymphocytes to KGla was adapted from the rat lymphocyte-lymph note b~nA1ng a6say which has been described by Stamper and Woodruff (1976) and Sackstein et al.(1988). Cytospin preparations of KGla or other cell lines were made on a Cytospin 3 Cytocentrifuge (chAnAo~ Lipshaw, Pittsburgh, PA).
Frozen rat LN sections 8~m thick were mounted on 6lides, and lymphocyte b~lA1 ng to LN HEV 6erved as a po6itive ~ ol in all experiment6. Slide6 were SUBSTITUTE SHEET (RULE 26) WO96/11012 PCT~S95113736 air dried, ~ixed in 3~ glutaraldehyde (Electron Microscopy Sciences, Fort Wa6hington, PA) in PBS, rin~ed with PBS, incubated in 0.2M L-ly6ine (Sigma Chemical Company, St. Louis, M0) to block unreacted glutaraldehyde, then rin6ed and held in RPMI 1640 with 1% FBS at 4C until u6e in experiment6.
Lymphocyte su6pensions (200~l) were overlaid onto cytospin or LN 6ection6 in duplicate and placed on a rotating platform (80rpm) at 4C
for 30 minutes. Slides were then rinsed in cold PBS to remove non-adherent lymphocytes, fixed in 3%
glutaraldehyde, and stAi n~A with methyl green-thionin. Slides were examined under the light microscope for adherence of lymphocytes to KGla or LN HEV.
Number of lymphocytes adherent to confluent area of KGla were counted by light microscopy using an oc~llAr grid under 250X
magnifi~ation. Quantitation wa6 performed by examining two fields per ~lide, minimum of two ~lides per experiment, three ~eparate experiment6.
Re6ult~ are pre6ented aG % b~ nA ~ ~g compared to corre6ponA~q untreated control 6ection6.
Treatment of Lymphocytes w~ th Potent ~ al Inhibltor6. Lymphocyte6 in RPMI 1640 medium with 5~ FBS were pre-1nc~h~ted (30 min on ice) and the a66ay performed in the prer~.nce of the following SUBSJITUTE SHEET (RULE 26) Wo96/11012 PCT~Sgs/13736 inhibitor6: lmN EDTA (no pre-incubation period); lO
mM D-mannose-6-phosphate (Sigma); lO ~g/ml PPNB
(kindly provided by Dr. M.E. Slodki, USDA, Peoria, IL); and 5 ~g/ml fucoidin (Sigma).
S An tibody Bl ock ~ ng Experimen ts .
Lymphocytes (l x 107 cells/ml) were pre-incubated on ice for 20 minute6 with mAbs at l.O ~g/ml and used in the binding assay without further washing.
The following ~Abs were used: LAM1-3 (anti-L-selectin) (kind gift of Dr. ~homas Tedder, Duke University, Durham, N.C., and also obtained from Coulter Corp., Hialeah, FL~; anti-CD45 (leukocyte Common Antigen) (Becton Dickinson, San Jose, CA);
and IgGl (i60type control) (Coulter). In 60me lS experiments, prepared KGla filides were incubated with 0.2 ~g of anti-CD34 Abs {HCPAa-l (clone MylO) and HPCA-2 (clone 8Gl2) (Becton Dickinson), QBENDlO
(AMAC) and 12.8 (kindly provided by Dr. Pat Roth, Coulter cor`p.)~ in RPNI 1640 with 5% FBS for 30 minutes prior to the b~n~ng as~ay.
PMA ~rreatment of Lymphocyte~.
Lymphocytes were suspended at l X 107 cells/ml in cell culture medium and ~n~llh~ted l hour at 37C
with or without lO ng/ml PMA (Gibco-BRL). Cell6 were then washed twice in PBS and used in either the lymphocyte b~nA~g a6say or analyzed for ~urface antigens by flow cytometry (see below).

SUB~IJUTE SHEET (RULE 26) WO96/11012 PCT~S95tl3736 Enzyme Treatment of ~Gla or LN. cytospin preps KGla or LN frozen sections were glutaraldehyde-fixed, then treated with various enzymes prior to the bi~A~ng assay. For treatment with neuraminidase (sialidase), 61ides were rin6ed twice with enzyme buffer (50 mM NaAc, 154 mM NaCl, 9 mM CaC12, pH 5.5), then inc~lh~ted 30 min. at 37C
with 50 ~1 of buffer (oonL.ol) or undiluted neuramin1AA~ (1.2 U/ml, Boehringer MAnn~e;m~
Tn~iAn-Apolis, IN). In protease studies, slides were incubated with RPMI 1640 alone or RPMI 1640 contAinin~ enzymes: 100 U/ml chymotrypsin (Sigma) (115 min. at 37C), or 0.1% bromelA;n (Sigma) (30 min. at 37C); to assess specificity, the protease inhibitors PMSF (1.0 mg/ml, Sigma) and chymostatin (900 ~g/ml, Boehringer MAnnhplm) were coinr~h~ted with chymotrypsin (100 U/ml) for 15 min. at 37C.
Following enzyme treatments, slides were washed X3 with RPMI 1640 and placed in RPNI 1640 with 1% FBS
untll use in the binA;n~ assay.
XGla cell~ in su6pension (4 x 107 cells/ml) were incllhAted with 0-sialogly~yLG~ein endopeptidase (Accurate Chemical and ScientifiC
Corp., Westbury, NY) (0.24 mg/ml, 37 C, 30 min.), washed X3 with 2~ FBS in PBS, and cytospin preparations were made for use in the b~nA~n~
a6say. To verify the activity of the enzyme, cells SVBSmLl~E SWEET (RULE 26) were tested for the cleavage of CD34 by flow cytometry using QBEND10 mAb.
Antigen Express~on by Flow Cytometry.
Flow cytometric analysi~ was performed using the following commercially-available mAbs together with isotype-matched control~: TQ1 (anti-L-selectin), LAM1-3 (Anti-L-selectin), 4B4 -(Anti-VLA-4) (all -from Coulter Corp.); QBEND10 (anti-CD34) (AMAC, Westbrook, NE); anti-CD44, LFA-1-~ (anti-CD18), LFA-l-~ (anti-CDlla), HPCAS-2 (anti-CD34), anti-CD45, Leukosialin (anti-CD43), anti-Sialyl-LeX (all from Becton Dickinson). Cells (1 X 106) in 100 ~l of PBS with 2% FBS were incubated on ice for 25 minute~ with Ab as per manufacturer's recommendations, wA~h~A X3 and analyzed on a FACStarP~Us (Becton Dickin~on).
Fluorescence Act~vated Cell Sort~ng of ~Gl~ cells. XGla cells were st~n~A with anti-CD34 mAbs ~QBE~10-FITC in two experiments, HPCA-2-PE in one experiment) and positive and negative expre~sing cell6 were sorted on a FAC~tarPLU8 flow cytometer equipped with an argon laser tuned at 488 nm (Becton Di~nr~n). Sorted cell population6 were rest~n^~ with anti-CD34 Ab directed at epitopes not used for ~orting and were analyzed to determine the efficiency of the sort. Cytospin preparat1ons were made of the positive and negative SUBSllME SHEET (R(~LE 26) W096/11012 PCT~S95/13736 sorted fraction6 and were used in the lymphocyte binding assay.
Transfection of C05-7 w~ th CD34 cDNA .
COS-7 cells were transiently transfected with human full-length CD34 cDNA in pCDM8 plasmid (a gift from Dr. Daniel Tenen, Boston, MA) using a DEAE Dextran transfection method (Selden, 1992). Briefly, COS-7 cells were incubated for 4 hours at 37C with lO ml of transfection solution contAining 20-40 ~g of plasmid DNA, 10% Nu Serum (Collaborative Biomedical Products, Bedford, MA), 400 ~g/ml DEAE Dextran (Sigma), and lO0 ~ chloroquine (Sigma) in D~llheccols Modified Eagles Medium (Gibcon-8RL).
Cells were then rinsed and treated with lO~ DMS0 lS (Sigma) in PBS for two minutes at room temperature, rinsed in PBS, and incubated in tissue culture media for 3 day6. In-one set of experiments, trypsinization was avoided by growing transfected cells directly on glass slides for subsequent use in the binding assay or for analysis of CD34 expres~ion by fluorescence microscopy. In other experiments, COS-7 cells grown on lO cm plates were removed with trypsin/EDTA (0.25%/lmM, Gibco-BRL), then analyzed for CD34 expression by flow cytometry. These trypsinl~ed cells were then placed on slides by cytospin for use in the lymphocyte b~n~ n~ a8~ay.

SUBS~llUTE ~HEET (RULE 26) CA 02202472 1997-04-ll W O96/11012 PCTrUS95/13736 . -29-B~AMPL~ 1 Lymphocytes Bind to RGla. Lymphocyte~
(both PBL and TDL) adhered specifically and reproducibly to KGla, ~ut not to RPMI 8402, HL60, Nalm 16, K562, or Raji cell lines in the in vitro binding assay (Table 1). All experiment6 were performed in parallel with LN frozen sections ar positive controls. Lymphocyte binding to RGla was observed under conditions identical to those whereby L-selectin mediates binding of lymphocytes to LN HEV.

Lymphocyte pin~ing to RGla is Med~ated by L-selectin. To directly examine whether lymphocyte attachment was mediated by L-selectin, PBL were pre-incubated with the anti-L-~electin mAb LAM1-3, anti-CD45, or IgG1 isotype cGnLlol Abs. The LAM1-3 Ab completely inhibited lymphocyte binding to KGla and LN ~on~.ol, while CD45 and isotype control mAbs did not affect binding (Fig. lA & lB). In order to quantify the relative amounts of Ab attachment to lymphocytes, Ab-treated lymphocytes were incllh~ted with goat-anti-mouse FITC-conjugated ~econAAry Ab and analyzed by flow cytometry. Although the amount of anti-CD45 Ab on lymphocytes was significantly greater than that of LAM1-3 a~
indicated by mean ahAnnel fluorerc~nae (Fig. 2), SUBSrmJTE SHEE~ (RULE 26J

WO96/11012 PCT~S95/13736 j LAMl-3 alone blocked lymphocyte adherence to KGla and LN HEV, indicating that this ef~ect was specific and not secondary to charge or 6teric alterations of the lymphocyte membrane.

The Effect of Enzyme Treatment of ~Gla on Lymphocyte B~ndin~. Pretreatment of both KGla and LN col.LLol sections with neuraminida6e (60 mU), chymotrypsin (lO0 U/ml) or bromelain (O.l~) prior to the binding a~ay abrogated binding of lymphocytes, while treatment with buffer or medium alone did not alter binding capacity. In addition, the effects of chymG~Ly~in were confirmed by coincubation with the protease inhibitors lS chymostatin and PMSF, which prevented chymotrypsin effectc on lymphocyte binA;ng. However, pretreatment of KGla with 0-6ialogly~0~LGLein endopeptidase had no effect on lymphocyte binA~ng despite complete enzymatic removal of the CD34 epitope recognized by QBENDlO mAb. (Table 2) Lymphocyte B~nd~ng to RGla ~ C~lcium Dependent. Lymphocyte b;nA~n~ to KGla and to LN
control ~ections was completely inhibited by the presence of EDTA, indicating a calcium requirement for lymphocyte-KGla b;n~ng.

SUBSTITIJTE SHEET (RULE 26) Wo96/11012 pcT~s9sll3736 M~nnose-6-Phosph~te, PPME, and Fucoidin Inhib~t Lymphocyte Binding to RGla. The specificity of lymphocyte-RGla binding wa~
investigated by treating PBL or TDL with carbohydrate inhibitors of L-6electin-HEV
interactions prior to the adherence assay. Man-6-P
(10 mM), PPME (10 ~g/ml), and fucoidin (5 ~g/ml) all inhibited lymphocyte binding to both KGla and LN control section6. (Table 2) PMA Treatment of Lymphocytes Results ~n the Loss of Binding to ~Gla. PBL were incubated for 1 hour at 37C with 10 ng/ml PNA, then used in the lymphocyte binding assay. PMA-treated PBL were unable to bind to either KGla or LN HEV, while control PBL demonstrated high amounts of b;n~in~.
(Table 2) Loss of surface L-selectin was a~sessed by flow cytometric analysis of TQl levels in ~oll~Lol and PMA-treated PBL. PNA-treated lymphocytes ~howed a dramatic decrease in TQl mean c~n~l fluorP~cence (to levels less than 10% of that of untreated cells) in three separate experiments. PMA-treated PBL were also analyzed for expression of CD44, LFA-l (both ~ and B
ChA~lF)., and VI,A-4, and expression of these SUBSrlTUI~ SHEEt (RULE 26) Wo96/11012 -32- PCT~S95/13736 .
adhesion mo~lecules following PMA exposure was identical to expre6sion on control PBL.

B~AMPLE 2 Pretreatment of ~Gla w~th Anti-CD34 Antibodies Did Not Inhi~t Adherence of Lymphocytes. Cyto~pin preps of KGla were preincubated with anti-CD34 Abs and the b~n~;n~
assay was performed in the presence of the Abs.
Monoclonal ABS to four different CD34 epitopes were used alone or in combination, including the clones MylO, QBENDlO, 8gl2, and 12.8, in amounts ranging from 0.2 to 17 ~g/slide. Anti-CD45 (irrelevant control) and IgGl (isotype control) Abs were also lS tested. None of the anti-CD34 Abs inhibited lymphocyte binding to KGla, despite immunohistochemical evidence of extensive Ab binding to the glutaraldehyde-fixed KGla sections.

Other Surface Antlgens on ~Gla do not Appe~r to Med~ate Bind~ng. The surface expression of several antigens on KGla, RPNI 8402, HL60, Nalm 16, K562, and Raji was analyzed by flow cytometry (Table l). LFA-l, FLA-4, CD44, Sialyl LeX, and CD43 were all expressed by KGla and at lea6t one other cell line that did not ~ rort lymphocyte adherence. Of note, although RPMI 8402 cells SUBST~TUI E SHEET (RULE 26) Wo96/11012 PCT~S95/13736 .
express CD34 at levels comparable to KGla, there was no adherence of lymphocytes to these cells in the binding assay.

CD34 Posttlve and Negative ~Gla Cells Supported Equivalent Amount~ of Lymphocyte Bind~ng.
CD34+ and CD34- KGla cell6 were separated by fluorescence activated cell sorting and cytospin preparations of each population were made. The ~n vitro adherence of lymphocytes was identical in the CD34~ and CD34- populations despite an enrichment of >90% and <10% CD34+ cell8 in the respective populations (Fig. 3 and 4; Table 2).

E~AMPLB 3 CD34-Transfected aos-7 Cells Did Not Support Lymphocyte Adherence. COS-7 cells were transfected with CD34 and tested in the ~n v~tro ~nA~ng assay, and both trypsinized and intact transfected COS-7 cell6 failed to ~ o~L
lymphocyte adherence. By flow cytometric analy6i6, transfected cells were a~oximately 60% po6itive for CD34 expre66ion, and the mean ch~nn fluorPFcPnce wa6 greater than that of KGla ~ul.LLol cells (Fig. 5). Intact, ~lLLy~sinized COS-7 cells transfected with CD34 also strongly expressed CD34 SUBSTI~UJE SHE~T (RVLE 26) W O96/11012 PCT~US95/13736 , (~90% positive a6 estimated by fluorescence microscopy)~
Throughout this application variou~
publications are referenced by citation or number.
Full citations for the publications referenced by number are listed below. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully de6cribe the ~tate of the art to which thi6 invention pertains.
The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.
Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be under~tood that within the scope of the app~AP~
claims, the invention may be practiced otherwi6e than a~ ~pecifically described.

SUBSrlTUT~ SHEET (RUIE 26J

WO 96/11012 PCTtUS95tl3736 .

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SUBSrlTlJTE SHEET (RULE 26~

wos6/11oi2 PCT~S95/13736 j T~ble 2. Lymphocyte A~herence to ~Gla LYMPHOCYTE TREAq~ENrNean (SEM) o~ Bindlng EDTA 0-3 (0 3) Mannose-6-P 5.7 (1-0) Fucoidin 1.4 (0-4 ?
PPME 5 4 (o 5) LAM1-3 mAb 1.9 (0.4) Anti--CD45 mAb 98.7 (6.3) IgGl Control mAb 115.1 (9.0) PMA 1.1 (0.3) XGlA TREATMENT:
Anti CD34 mABst 116.2 (7.7) Anti CD45 mAb 98.0 (3.6) IgGl Control mAb 101.8 (8.5) CD34-Positive Sort 102.8 (3.5) CD34-Negative Sort 104.1 (4.2) Neuraminidase 3.1 (0-7) Neuraminidase Buffer Co.l~ol100.5 (6.7) O-Sialogly~o~ro~ein Endopeptidase98.4 (2.3) Bromelain 3.8 (0.4) ChymG~ in 6.7 (0.7) ChymG~ in, PMSF, Chymo6tatin94.0 (3-8) tCombination o~ HPCA-l, HPCA-2, 12.8 and QBEND10 mAbs.

SU~SrlTU~E SHEET (RULE 26) WO96/11012 PCT~S95/13736 j ~F~R~

Abdullah KM, Udoh EA, Shewen PE, Mellors A: A
neutral glycoprotease of Pasteurella haemolytica A1 specifically cleaves O-sialoglycoproteins. Infect Immun 60:56, 1992.

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SUBSrl~E SHEFr (RULE 26~

.

W O96/11012 PCTnUS95/13736 Gowans, JL, Knight EJ: The route of re-circulation of lymphocytes in the rat. Proc Roy Soc Lond B
159:257, 1964.

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SUB~TITUTE SHEET (RULE 26) WO96/11012 PCT~S95/13736 Kobayashi M, Imamura M, Uede T, Sakurada K, Maeda S, Iwasaki H, Tsuda Y, Musashi M, Miyazaki T:
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SU~SrIME SHEE~ (RULE 26i~

WO96/11012 PCT~S95/13736 Majdic O, Stockl J, Pickl WF, Bohuslav J, Strobl H, Scheinecker C, Stockinger H, Knapp W: Signaling and induction of enhanced cytoadhesiveness via the hematopoietic progenitor cell surface molecule CD34. Blood 83:1226, 1994.

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SUBS~1ME SHEET (RVLE 26) .

WO96/11012 PCT~S95/13736 Reinherz EL, Morimoto C, Fitzgerald KA, Hussey RE, Daley JF, Schlossman SF: Heterogeneity of human T4 inducer T cells defined by a monoclonal antibody that delineates two functional subpopulations. J
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SUBSnME SHEET (RUEE 26) .

WO96/11012 PCT~S95/13736 -~5-j Tedder TF, Penta AC, Levine HB, Freedman AS:
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ITUTE SHEET (RULE 26~

WO96111012 PCT~S95/13736 Woodruff JJ, Katz IM, Lucas LE, Stamper HB: An in vitro model of lymphocyte homing II. Membrane and cytoplasmic events involved in lymphocyte adherence to specialized high-endothelial venules of lymph nodes. J Immunol ll9:1603, 1977.

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SUBSTI~lJTE SHEET (RllLE 26~

. .

Claims (16)

What is claimed is:

1. An isolated and purified glycoprotein and functional analogs thereof characterized by (a) being expressed on at least primitive hematopoietic cells, (b) being a ligand for L-selectin, the binding of ligand to L-selectin not being inhibited by anti-CD34 antibodies; and (c) not being identified by MECA-79 a monoclonal antibody which identifies ligands of L-selectin on lymph node high endothelial venules.

2. An isolated and purified glycoprotein as set forth in claim 1 wherein said glycoprotein is a membrane-associated glycoprotein.

3. An isolated and purified glycoprotein as set forth in claim 1 wherein said glycoprotein functions as an adhesion protein ligand.

4. An isolated and purified glycoprotein as set forth in claim 1 wherein said glycoprotein facilitates attachment of lymphocytes to hematopoietic cells.

5. At least one antibody directed against said glycoprotein as set forth in claim 1.

6. An antibody as set forth in claim 5 wherein said antibody is a monoclonal antibody.

7. A method of targeting cells expressing the glycoprotein as set forth in claim 1 including the steps of preparing a monoclonal antibody directed against the glycoprotein as set forth in claim 1, preparing an immunotoxin utilizng the antibody, exposing a population of cells to said antibodies, and killing cells bound to the immunotoxin.

8. The method of claim 7 wherein the toxin is selected from the group consisting of ricin A chain, pseudomonas exotoxin A, diphtheria toxin and chemotherapeutic compounds.

9. The method of claim 7 wherein the cells are exposed to the immunotoxin in vivo.

10. The method of claim 7 further characterized by the cells being selected from the group consisting of leukemic cells, malignant hemopoietic progenitor cells and other malignant cells expressing the glycoprotein.

11. A method of selecting for cells expressing the glycoprotein as set forth in claim 1 including the steps of preparing an antibody directed against the glycoprotein as set forth in claim 1, exposing a population of cells to said antibodies, and selecting cells bound to the antibody.

12. A method of selecting against cells expression the glycoprotein as set forth in claim 1 including the steps of preparing an antibody directed against the glycoprotein as set forth in claim 1, exposing a population of cells to said antibodies, and removing cells bound to the antibody.

13. The method of claim 12 wherein said removing step is selected from complement-mediated lysis, panning, cell sorting.

14. A method of regulating hematopoiesis including the steps of selecting cells with an appropriate level of expression of the glycoprotein as set forth in claim 1 from a patient, culturing the selected cells, and reinfusing the patient with the expanded selected cell population.

15. A method of regulating inflammatory response by interrupting cellular migration into lymph nodes and sites of chronic inflammation including the step of administering to a patient antibody directed against the glycoprotein as set forth in claim 1.

16. The method of claim 15 further characterized by the inflammatory response being as found in the group selected from autoimmune disorders, post-ischemic tissue injury and sepsis.