CA2289727A1 - Protein smi on hematopoietic stem cells - Google Patents

Abstract

A protein designated "SM1" has a molecular weight of about 230 kDa protein, as measured by immunoprecipitation and SDS-PAGE. SM1 proteins are present on the surface of human and mouse hematopoietic stem cells, respectively, and on primitive progenitor cells, but are absent from the surfaces of other cells, such as FDC-P1 myeloid progenitor cells, EL4 T-cells, WEHI-3 myelomonocytic cells, and 70Z/3 pre-B lymphoid cells, or from differentiated hematopoietic cells of human cord blood or mouse bone marrow. Anti-SM1 antibody can be employed in making a preparation that is enriched for hematopoietic stem cells.

Description

PROTEIN SMl ON HEMATOPOIETIC STEM CELLS
BACKGROUND OF THE INVENTION
The present invention relates to a protein, referred to here as "SMl," in a form that is substantially purified from other proteins . With a molecular weight of about 230 kDa as measured by immunoprecipitation and SDS-PAGE, SM1 is present on the surface of human and mouse hematopoietic stem cells and primitive progenitor cells, but is absent from those of other cells including FDC-P1 myeloid progenitor cells, EL4 T-cells, WEHI-3 myelomonocytic cells, and 70Z/3 pre-B lymphoid cells, or from differentiated hematopoietic cells of human cord blood or mouse bone marrow. The present invention further relates to methods of using anti-SM1 antibody to produce an enriched hematopoietic stem cell population.

All circulating blood cells develop from pluripotent 1J stem cells through the process of hematopoiesis.

Hematopoietic stem cells are undifferentiated cells capable of self-renewal and differentiation into committed progenitor cells of the myeloid, erythroid, megakaryocytic and lymphoid blood cell lineages. A

thorough analysis of hematopoietic stem cells is fundamental to a comprehensive understanding of the developmental biology of the hematolymphoid system.

Relatively little is known, however, about hematopoietic stem cells.

Functionally, hematopoietic stem cells are capable of long-term reconstitution of the hematolymphoid system of lethally-irradiated recipients in vivo. Spangrude Johnson, PNAS 87:7433-7437 (1990); Spangrude et al., Blood 78:1395-1402 (1991). They also can differentiate into short-term hematopoietic stem cells, called day 12 spleen colony-forming units (CFU-S), which can be observed in in vivo assays for spleen foci formation.

Spangrude et al., Science 241:58-62 (1988); Molineux et al., Exp. Hematol. 14:710 (1986); Nakahata & Ogawa, PNAS

79:3843-3847 (1982). In addition, another property of hematopoietic stem cells develop a "cobblestone"

morphology upon adherence in vitro to a layer of stromal cells. Wong et al., Immunity 1:571-583 (1994).

Efforts to characterize hematopoietic stem cells in more detail have been hampered primarily because of the proportionately minute amount (less than 0.01%) of hematopoietic stem cells as compared with all cells , even in blood cell-forming organs such as bone marrow or the fetal liver. Li & Johnson, Blood 85:1472-1479 (1995).

Accordingly, the elucidation of physical characteristics unique to hematopoietic stem cells is desirable as a means to produce enriched stem cell populations. For example, see Spangrude et al., Blood 78:1395-1402 (1991).

All known hematopoietic stem cell enrichment protocols involve cell-separation methods based mostly on the selection for cell surface markers or other physical means, such as density gradient centrifugation, counter flow centrifugal elutriation, and cell sorting based on light scattering properties. Bertoncello et al., Expt.

Hematol. 13:999-1006 (1985); Mulder & Vi~sser, Expt.

Hematol. 15:99-106 (1987); Ploemacher & Brons, F,xpt.

Hematol. 17:263-271 (1989); Szilvassy et al., PNAS

86:8798-8802 (1989). Although methods of producing enriched populations of hematopoietic stem cells have been described, the absence of unique markers has precluded the isolation of an unequivocally pure population of hematopoietic stem cells.

Some hematopoietic stem cells express cell surface differentiation antigen (Thy-1) and stem cell antigen-1 (Sca-1). They do not, however, express the lineage markers (Lin) characteristic of B cells (B220), granulocytes (Gr-~), myelomonocytic cells (Mac-1) and T

cells (CD4, CD8)). Spangrude et al., supra. The reportedly most ~ridely used hematopoietic stem cell -- enrichment protocol involves the use of monoclonal antibodies against Thy-i and Sca-1. Orlic et al., supra.

Only a subset, however, of Thy-1+, Sca-It and Lin- cells are able to repopulate lethally-irradiated recipients long-term. Smith et al., PNAS 88:2788-2792 (1991).

Selection based on Thy-1 and Sca-~ expression thus does not produce a pure hematopoietic stem cells population.

Similarly, other hematopoietic stem cell enrichment techniques such as those which involve the use of monoclonal antibodies against protein tyrosine kinases such as the W locus gene product, c-kit, and fetal liver kinase-2 (flk-2) apparently are unable to distinguish between hematopoietic stem cells and progenitor cells.

See, for example, Matthews et al., Cell 65:1143-1152 ( 1991 ) .

Another example of a cell surface marker associated with hematopoietic stem cells is CD34. A membrane phosphoglycoprotein, CD34 exists on hematopoietic stem cells, committed progenitor cells of all hematopoietic cell lineages, early multipotent hematopoietic progenitor cells, and endothelial cells. Krause et al., Hlood 87:1 (1996). CD34+ cells have been estimated to be about 2.5%

of total bone marrow cells, Osawa et a1 . , Science 273 :242 (1996), and 1-4% in humans and baboons. Civin et al., J.

Immunol. 133:157 (1984); Civin et al., Exp. Hematol.

15:10 (1987); Berenson et al., J. Clin. Invest. 81:951 (1988) .

Hematopoietic stem cells have been estimated to constitute less than 0.1% of total bone marrow cells.

Thus, selection based on CD34 alone does not yield a pure population of true hematopoietic stem cells. CD34 has been targeted in combination with other cell surface markers for stem cell purification. These markers include the so-called lineage-specific antigens, such as 3LA-DR, Thy-1, CD33, NB7R-1, c-kit, CD45 and CD38.

Sutherland et al., Blood 74:1563 (1989); Sutherland et al., Blood 78:666 (1991); Lansdorp et al., J. Fxp. Med.

172:363 (1990); Baum et al., PNAS 89:2804 (1992);

3riddell et al., Blood 79:3159 (1992); Drach et al., Blood 78:30 (1992); Gore et al., Blood 77:1681 (1991);

3riffin et al., Blood 60:30 (1982); Verfaillie et al., Exp. Med. 172:509 (1990); Terstappen et al., Blood 77:1218 (1991); Huang ~ Terstappen, Nature 360:709 !1992); Huang s~Terstappen, Blood 83:1515 (1994); Cardoso et al., PNAS 90:8707 (1993); Issaad et al., Blood 81:2916 (1993); Srour et al., J. Immunol. 148:815 (1992). Using such combinations, CD34t/CD38~ cells were found to comprise less than O.lo of total human bone marrow cells, ~ivin et al., Blood 88:4102 (1996), and CD34+Thy-1+Liri cells to comprise 0.05% to 0.1% of human fetal bone :narrow cells . Baum et a1. , PNAS 89 :2804 ( 1992 ) .

Fractions of CD34+ cells, enriched by selection for CD34 alone or in combination with other markers, have been found to exhibit primitive progenitor or stem cell functions. In vivo studies have been performed in mice, Wong et al., supra, to assess the ability of ~D34-enriched cells for long-term reconstitution, a defining characteristic of hematopoietic stem cells.

With respect to human cells, a number of in vitro assays nave been employed to detect properties typical of true hematopoietic stem cells. These assays include those that examine primitive multi-lineage hematopoietic progenitor/stem cells, Brandt et al., Blood 83:1507 (1994); Rusten et al., Blood 84:1473 (1994), high proliferative potential cells, Muench et al., Blood 83:3170 (1994) , blast-colony forming cells, Leary & Ogawa Blood 69:953-956 (1987), cobblestone-forming cells, Henschler et al., Blood 84:2898 (1994), and long-term culture initiating cells (LTC-IC), Lemieux et al., Blood 86:1339 (1995); Verfaillie & Miller, loc. cit. 84:1442 (1994). Although these primitive cells do exhibit certain properties associated with rematopoietic stem cells, such as high proliferative capacity and the ability to differentiate into various lineages of hematopoietic cells, arguably CD34-enriched cells do not a constitute a pure population of true hematopoietic stem cells. Lord & Dexter, Expt. F~ematol. 23:1237 (1995).

Nonetheless, the CD34-enriched population of cells has been shown to have high clinical value. Emerson, supra.

The recent establishment of a cell line from a lethally-irradiated recipient mouse reconstituted with fetal liver cells previously transduced with a rearranged retroviral genome has been reported. along et a3., supra.

BL3 cells exhibit all of the functional hematopoietic stem cell properties, i.e., they can reconstitute lethally-irradiated recipients long-term, they give rise to pre-CFU-S and colony-forming cells and they develop "cobblestones" upon association with stromal cells. In addition to being Thy-1+, Sca-1+ and Lin~, BL3 cells also express a transcription factor, GATA-1, known to be expressed in hematopoietic stem cells. Sposi et al., PNAS 89:6353-6357 (1992).- Furthermore, BL3 cells are embryonic in origin, having derived from fetal liver cells of 12-day old mouse embryos. HL3 cells thus may possess different cell surface markers than adult hematopoietic stem cells. Jordan et al., supra;

Spangrude et al., supra.

The foregoing discussion highlights a need for other cell surface markers, identified on hematopoietic stem cells, specifically to enable the production of more highly enriched hematopoietic stem cell populations, and generally to facilitate a better understanding of the growth and differentiation of immature blood cells.

SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to identify and isolate a marker that is present on the cell surface of human and mouse hematopoietic stem cells and primitive progenitor cells, but that is not present on committed progenitor cells or mature blood cells. It also is an object of the invention to provide for the use of such a marker to identify putative _~ematopoietic stem cell regulatory factors. It is a further object of the present invention to provide an antibody against a cell surface marker from human or mouse hematopoietic stem cells or primitive progenitor cells that can be employed to produce an enriched hematopoietic stem cell population.

In achieving these and other obj ectives , the present inventors have provided SMl protein substantially purified from other proteins, where SM1 has a molecular weight of about 230 kDa, as measured by immunoprecipitation and SDS-PAGE, is present on the surface of human and mouse hematopoietic stem cells and primitive progenitor cells, but is absent from the surface of other cells, such as FDC-P1 myeloid progenitor cells, EL4 T-cells, WEHI-3 myelomonocytic cells, and 70Z/3 pre-B lymphoid cells, or from differentiated hematopoietic cells of human cord blood or mouse bone marrow. The objectives also are achieved by an antibody against SM1 and the use of the antibody to enrich for hematopoietic stem cells.

Pursuant to one embodiment, anti-SM1 antibody is used to prepare a composition enriched for hematopoietic stem cells according to the invention. The inventive methodology comprises the steps of (a) providing antibody that binds SM1, (b) immobilizing the antibody on a support platform such that the antibody retains its SM1-binding capability, then (c) bringing a mixed population of cells containing putative hematopoietic stem cells into contact with the antibody such that the stem cells adhere to the support platform, and !d) removing nonadherent cells, whereby a population enriched for hematopoietic stem cells remains adhered to ~he support platform.

Another embodiment of the invention is a kit for preparing a composition enriched for hematopoietic stem cells, comprising (i) an antibody that binds SM1 and ;ii) written directions for the use of the kit to effect antibody-facilitated enrichment for hematopoietic cells of high purity capable of effecting long term hematopoietic reconstitution. For purification of hematopoietic stem cells to be used for human treatment, e.g., in a transplantation context, bone marrow can be obtained from a HLA-identical or nearly identical donor.

Bone marrow cells can then be contacted with the antibodies of the kit. Cells isolated in this manner may be subjected to growth factors and cytokines to achieve a sufficiently pure population of hematopoietic stem cells suitable for transplantation into human patients.

In a further embodiment of the present invention, a methodology for detecting in a sample a hematopoietic factor that binds SM1 comprises (a) contacting a sample suspected of containing said growth factor with labeled-SM1, and ib) detecting the binding of the hematopoietic factor with labeled-SM1.

Another embodiment of the invention relates to a kit for the detection of a hematopoietic factor that binds SM1, comprising labeled-SM1, and further comprising written instructions for the use of the kit.

One other embodiment of the invention includes methods of amplifying or expanding in vitro human SM1 cells. With respect to growth in a liquid culture system, SM1 cells may be suspended in liquid media with additional growth factors and cytokines. In a stromal coculture system, SM1 cells may be grown on or within an _ g _ adherent layer of mixed stroma cell preparations, with or iaithout the addition of growth factors and cytokines.
Yet another invention provides embodiment an of the isolated DNA SM1. A particular molecule encoding embodiment of the invention provides an isolated DNA

molecule that the following nucleotide sequence includes (SEQ. ID. N o. 1):

GGAATTCCGN CAGCAAGTTC TTATTCTGCC GTGATTCAGC
TAAGAATTTT

ACAAAGAGGG GAAAGCAGTT GAA.AAAGAGA TCAGCAGAAA
TAGCAGCACC

GGCCCAGAGC ATTGCTCACC TGGCCCACAG CGTGTTCCTT
ACAAGCGCTA

AGTGTCTGTT CCTGTCACCT CTGTGTCTAC CCAACTGCCT AATACAGTTC

TCAGTAAGAC AAGTACACCT TCATCAAATG TGAGTGCTAG ATCACAGCCT

TTGTCTCCTG TAGCCTCTGT AAGTAATGCA TTAACATCAC CAGTTAAGAC

TAGCCAAAGT GAAGCAGGAA AAGTCAAGAG TACCGCTTCA TCCACCACAC

TCCCCCAGCC TCACACTTCA CCTACCATTT CATCAACAGT TCAGCCTCTC

TTGCCAGCAA CAACACTAAA TGAATCTACA GATCCTGGCA GTTCCATCCC

CTGTTTTTCA CAGCAAACTG TTGATTCTTC TGAGGCAAAG CAAGAACTAA

AAACTGTATG TATACGAGAT TCACAGTCAA TTCTTGTTAG GACTCCAGGT

GGGAACACTG GAGTTGTAAA AGTACAAACT AATCCGGAAC AAAATTCACC

CAACAGTTTA TCTTCAAGTT CTGTTTTCAC CTT'fACACCTCAATTTCAGG

CATTTCTTGT GCCAAAATCA ACATCATGCT CTGCTTCCTC ACAAGTAGCC

GGAGTGACTA CTACATCTAG TCTACCATCT TTCAGCCAAG CAATCTACGT

NTGTGTNGCT TCATCCACCC ATGGGAAAAA TCTCAAATCT ACACAAGGCC

AAACCTTGAG CAGTGGTATG TAGGCCCCAT GATAGAAAAP.ACGTCATACA

TCAACAACAA ATAGTTCAGT GAGTGTAATT AGCATATCAA CAGGAAATNN

NGGGCAAACC AATACAAATG TTATTCATAC ATCAACTAAA CCACAACAAG

TAGATTGTAT CACNAAAAGT TACCCAGTTA CAAGATCAGA AGCAACAACA

GCAGTAAATG GTGATGTGCT CGGTGAGACT CCAGGTCAGA AACTGATGCT

GGTGTCAGCT CCATCTGGTC TCCCTTCTGG CAGTGTACCT TCAGTTAACA

CGGCACCAGA ACCGACATCT GCAGGTGTGT CTACCCAGAA GGTAGTTTTT

ATTAATGCTC CAGTTCCTGG TGGCGCTTCA TCCTCAGCTA TTGTTGCAGA

ATCATTAAGA CAGTCACTTC CTTCTCCCAC AAATACTGTA TTACTAGTGT

GCTTGTAGTA GTTAACTCCA CCATCTTTGT AAGCTAATGA AATTGTGAGT

CACCCATTTA TATCTTAATT TTTAATCATG TCAGTTCTTG AATGGGTATC

TCCTTAGCCT GCTGATTTCT AAAGAAAGTG GGTGGAGAAA
TTTTCTTTCT

_ g _ TTAATTTAGA CGTTTGTTTG CAATAAAA.AGAATTC

Yet another particular embodiment of the invention provides an DNA molecule isolated that includes the following nucleotide equence 2):
s (SEQ. ID.
No.

GAATTCTTTT TATTGC_~AACAAACGTCTAA ATTAATTTCT CCACCCACTT

TCTTTAGAAA GAAAAAGAAA TCAGCAGGCT AAGGAGATAC CCATTCAAGA

ACTGACATGA TTAAAAATTA AGATATAAAT NGGTGACTCA CAATTTCATT

AGCTTACAAA GATGGTGGAG TTAACTACTA CAAGCACACT AGTTATACAG

TATTTTGTGG GAGAAGGGCA TACAGACATG GCTAACTTCA TATAGATCCC

ATTAGACAAC '_"GGATTTACAACAAGTTTTT TTAATAAGAA ATGGGCAAAG

CAGCTTTCTT TTCAGAATCA AAATGCAGAA CAAATGGAAA AATTATGGTA

TTAACCTTCA CAAGTTTGAG CCTCCACAAA TAATGCAACC AAGTTTTACA

TTTTTAACAG CCCTTCTACA TACACTCCAT CTTCTCTATC TTAGTTCCAA

GTTTTAGTTT TCAATCCCAA TTATACCAAT TCCATTGTTA TTTTAAGAAA

TACNNAGCAA ACTACAGAGA GGATGGAGTG TAATATGAGC AGTACAGAGT

CTTAATGCAA TTCATGAGGA CCACTTAGTC CTTACATGAA TCTGGTTGCT

AACATTTCTA TTATATTGTG ACAATGACTC CCGACTGTTA TTCTCTGTGA

GAAATGGGGG GAGTAAATTC TTAATAAAAG ACACCAGGTA CAAAGCAACA

TTTTACTTCT GTTGTGATAA AAAAAAAAAA AGGTCACATT TTCAGATAAA

ATGTGGAACC CTGAAATCTG ACACATTCTC TTATCGTGCC ACCAATGCTG

AGGTTCTCTT ACGATTCACT TTTAAACTGC AATTAAAAAT GTACAAAAAA

GAAAAGAAAA AAANTCAACC CACAAAGCTT CTAAAAAAGG AACCCGCAGG

CACTTCCTCT TGTGGAATGT TTAAAA.AGTTAGCCTACTAA AGAAAACAGT

2 5 CGACTTCTTG TGAAGGTTTT GGAGAAATAT GTATCAGTTC GTT"I'TATTTG

GGTATTCAAT AATATCCTTG GTGATAATGC TGACTCCATG GCTTCTGACC

CCAGAATTGA CCCTGCTGCC ACTGGTTGTA GCCCTGAGAT TGATTTTTGT

AGCCACGATT GTTTCCTCGT CCTCTGAAGT TCTGGTTGTA GTTCCCTCTG

TTGGGCATTC CACCTCTGTT GTAGTTCCCT CTGTTTGAGT AACTACCACG

GCCAGGAAAA ACAGGGGCAC GAGGGTATGG ATAGCCGATT CCACCACTTC

CTCCACCGCC ACCACCTCTC TGTGGCATGT TGCCCTCCTA TTATATCCGC

CACGATTCCC AGGGGCTCCT CCTCTGAAAT TTCCACCACG CATATTGAAT

CCTCCACGTC TCTATGGCCA CCACCTCTGT TAAACTGGTT CTTGCCACTC

TTATTTTTAT TGCTTTTCTT TGAGCCAGTG TTCTGTTTCT TTTCTGGTGG

AAGAGCCTTT TTGCTTTCTT CCTTATATTG CTCCAAGAGT TTTTGGGCTT

CTTCCTTCTG AAGGGCAACA TAGGTTATTT CATCAAAGCA CTCAGCTACC

TCTGGGAGGG TAAAGTTTCC TTTCATTT

Other objects, features and advantages of the present invention will become apparent from the following detailed description. T_t should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTIONS OF THE FIGURES
Figure 1. DNA sequence of mouse SM1 gene.
Figure 2. Immunoprecipitation of SM1 surface protein specifically on BL3 cells. Immunoprecipitation of 35S-methionine labeled cells with SM1 antibody indicates that of the tested samples, only HL3 cells expressed SM1 protein on the cell surface.
Figure 3. Southern blot analysis on CFU-S DNA from recipients of 100 and 1,000 SM1+ cells. SM1+ cells in the mouse bone marrow was estimated initially to be about 10%. To investigate whether hematopoietic stem cells reside in a subset of SM1+ cells, cells were depleted that were positive for lineage specific markers, i.e., CD4 (T helper cells), CD8 (T killer cells), Gr-1 (granulocytes), TER119 (erythroid cells), Mac-1 (macrophages) and H220 (pre B cells). These Liri cells (for lineage negative) were further divided into SM1+ and SM1- cells. FAGS analysis was performed on mouse bone marrow cells by using PE (polyerythrin) conjugated antibodies directed against all the lineage specific markers and FITC-conjugated SM1 antibody. Figure 6 indicates the result of such a two-color analysis.
Figure 4. Two color-fluorescence activated cell sorter (FRCS) analysis of mouse bone marrow cells. SM1+
cells from mouse bone marrow was estimated initially to be about 10%. To investigate whether hematopoietic stem cells reside in a subset of SM1' cells, we depleted cells that were positive with lineage specific markers, i.e., CD4 !T helper cells), CD8 (T killer cells), Gr-1 (granulocytes), TER119 (erythroid cells), Mac-1 (macrophages) and H220 (pre B cells). These Lin- cells (for lineage negative) were further divided into SM1+ and SMl- cells. FACS analysis was performed on mouse bone marrow cells by using PE (polyerythrin) conjugated -0 antibodies directed against all the lineage specific markers and FITC-conjugated SM1 antibody.
Figure 5. Nucleotide sequence of a human SM1 gene.
Figure 6. FRCS analysis of human cord blood cells, double stained with PE-conjugated lineage specific 15 antibodies and FITC-conjugated anti-SM1 antibodies.
DETAILED DESCRIPTION OF PREFERRED Eb~ODIMENTS
A protein (SM1) has been discovered and substantially purified from other proteins. It has a molecular weight of about 230 kDa, as measured by 20 immunoprecipitation and SDS-PAGE. SM1 is present on the surface of human and mouse hematopoietic stem cells and primitive progenitor cells, but absent from the surfaces of other cells, including FDC-P1 myeloid progenitor cells, EL4 T-cells, WEHI-3 myelomonocytic cells, and 25 70Z/3 pre-B lymphoid cells, or from differentiated hematopoietic cells of human cord blood or mouse bone marrow.
Antibody Against SM1 In one embodiment, the present invention relates to 30 antibodies against SM1. In addition to their use for the enrichment for hematopoietic stem cells, such antibodies could represent research and diagnostic tools in the study of hematopoietic factors and the development of antibody conjugated therapeutic agents for the treatment ?5 of diseases. In addition, pharmaceutical compositions comprising antibodies against SM1 may represent effective therapeutics. Antibodies of the invention include polyclonal antibodies, monoclonal antibodies, and fragments of polyclonal and monoclonal antibodies.

The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et a1 . , Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992), which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., ANTIBODIES: A LABORATORY

MANUAL, page 726 (Cold Spring Harbor Pub. 1988) , which are hereby incorporated by reference. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG), in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana Press 1992). Methods of in vitro and in vivo multiplication of monoclonal antibodies are well-known to those skilled in the art.

Multiplication in ~itro may be carried out in suitable culture media such as Dulbecco~s Modified Eagle Medium or RPMI i64G medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, cr in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.

After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.

Therapeutic applications are conceivable for the antibodies of the present invention. For example, antibodies of the present invention may also be derived from subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., International Patent Publication WO 91/11465 (1991), and Losman et al., Int. J. Cancer 46:310 (1990), the respective contents of which are hereby incorporated by reference.

Alternatively, a therapeutically useful anti-SM1 antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning S murine immunoglobulin variable domains are described, for example, by Orlandi et al., PNAS 86:3833 (1989), which is hereby incorporated in its entirety by reference.

Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:

522 (1986); Riechmann et al., Nature 332: 323 (1988);

Verhoeyen et al., Science 239: 1534 (1988); Carter et al., PNAS 89: 4285 (1992); Sandhu, Crit. Rev. Biotech.

12: 437 (1992); and Singer et al., J. Immunol. 150: 2844 (1993), the respective contents of these publications are hereby incorporated by reference.

Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al., METHODS : A COMPANION TO METHODS IN ENZYMOLOGY , VOL . 2 , page 119 (1991); Winter et al., Ann. Rev. Immunol. 12:

433 (1994), which are hereby incorporated by reference.

Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).

In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered~~ to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Mature 368:856 (1994); and Taylor et al., Int. Immunol.

.:579 (1994) , which are hereby incorporated by reference.

Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.

Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.

For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups i5 resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S.

patents No. 4,036,945 and No. 4,331,647, and references contained therein. These patents are hereby incorporated in their entireties by reference. See also Nisonhoff et al., Arch. Hiochem. Hiophys. 89:230 (1960); Porter, 8iochem. J. 73:119 (1959); Edelman et al., METHODS IN

ENZYMOLOGY, VOL. 1, page 422 (Academic Press 1967); and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

For example, Fv fragments comprise an association of VH and V~ chains. This association may be noncovalent, as described in Inbar et al., PNAS 69:2659 (1972).

Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraidehyde. See, e.g., Sandhu, supra. Preferably, the Fv fragments comprise VH and VL
chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL.
2, page 97 (1991); Bird et al., Science 242:423-426 (1988); Ladner et al., U.S. patent No. 4,946,778; Pack et al., Hio/Technology 11: 1271-77 (1993); and Sandhu, supra.
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL.
2, page 106 (1991).
The isolation and characterization of SM1 protein was achieved through the establishment of a monoclonal antibody against SM1. To prepare specific monoclonal antibodies, a general procedure as described in Harlow &
Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratories (1988)), which is incorporated herein by reference. Three male Lew/hsd rats (Animal Center of Fox Chase Cancer Institute, Philadelphia, PA) were each immunized subcutaneously by injecting 5x10' BL3 cells _ 17 _ suspended in 0.5 ml PBS mixed with complete Freund's adjuvant. Pre-immune sera were collected prior to the injection. Three weeks 'ester, the rats were boosted subcutaneously with a dose of 1x108 BL3 cells. Three subsequent boosting injections were done at 2-week intervals. Immune antisera were collected after the second and third boosting injections and were tested by live-cell enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP). All sera were tested positive on BL3 cells and negative on EL4 cells (a T cell line).

The titer of the antisera ranged from 1:1,000 to 1:10,000.

Three days before fusion, another 100 million HL3 cells, with no adjuvant, were injected intravenously into one positive rat. On the third day, the rat was sacrificed by carbon dioxide asphyxiation, its spleen was removed, and a single cell suspension was prepared in Dulbecco's Modified Eagle Medium (DMEM) + 2% fetal calf serum (FCS). Splenic cells and YB2/0 myeloma cells were mixed at a ratio of 10 to 1 and fused in the presence of 50% polyethylene glycol (PEG). Hybridoma cell clones were selected by culturing the cell mixture in HAT

selection medium.

About two weeks later, the hybridomas were screened for the production of antibody specific for BL3 cells.

Indirect immunofluorescent labeling was employed by a standard procedure known in the art. One million washed BL3 cells or other control cells, such as EL4, FDC-P1 and WEHI-3 cells, were incubated with 80 ~,1 of hybridoma supernatant at 4C for 30 minutes, and after washing twice were further labeled with FITC-conjugated goat anti-rat IgG+M secondary antibody under the same conditions. After washing, the cells of each clone were then screened by light microscopic examination. The antiserum was used as a positive control and pre-immune serum or some hybridoma supernatants were used as negative controls. Cells from three out of 170 hybridomas were shown to be specific for BL3 cells. Of these three, one recognized a molecule designated SM1.

By a standard immunodiffusion assay, the SM1 monoclonal antibody has been shown to be an immunoglobulin IgM

allotype.

SM1 DNA Isolation The isolation of SM1 cDNA was performed by first constructing a Lambda gtll cDNA phage expression library.

The construction of the cDNA library was done as follows.

To isolate poly(A) RNA, total RNA was extracted using phenol/chloroform/Guanidine thiocyanate method. Sambrook et al., MOLECULAR CLONING 2nd ed. (Cold Spring Harbor Laboratory Press 1989). Cells (5x108 to 10x108) were lysed in 10 ml of 4M GTC solution (25 mM sodium citrate, 85 mM sodium lauryl sarcosine, 4M Guanidine thiocyanate and 0.1 M 2-mercaptoethanol). DNA was sheared by passing through an 20 Gauge needle. The volume was increased to ml by adding 10 ml of 4M GTC solution. 2 ml of 4M NaAc (pH 4.0) was added and mixed well before equal volume of 20 DEPC-HZO saturated-phenol was added. After the mixture was mixed thoroughly, 10% of final volume of chloroform was added and mixed vigorously again. The mixture was allowed to sit on ice for 15 minutes, and then centrifuged for 20 minutes at 2500 g (5000 rpm in Sorvall RC-5B centrifuge with Sorvall SA600 rotor). The top aqueous phase containing RNA was transferred to a new tube. An equal volume of isopropanol was used to precipitate RNA at -20C for 1 hour. An RNA pellet was obtained after centrifugation at 2500 g for 20 minutes and dissolved in 0.4 ml of 4M GTC solution. The RNA was precipitated again with 10' ~,1 of 1M HAc and 300 ~cl of ethanol. The final RNA pellet was dissolved in 0.5 ml of 1mM EDTA/0.05% SDS and stored at -70C. Poly(A) RNA was selected by passing through two rounds over an oligo dT-cellulose column from Collaborative Research.

Maniatis et al., MOLECULAR CLONING -- A LABORATORY MANUAL
(Cold Spring Harbor Laboratory, 1982). The 1X binding buffer consists of 20 mM sodium phosphate and 0.5 M NaCl.

The amount of poly(A) RNA selected was about 5% of total RNA applied with a ratio of O.D.~bo/O.D.~BO of 2Ø The poiy(A) RNA was aliquoted, mixed with one tenth volume of 3M NaAc and three times volume of ethanol, and stored at -70C.

To initiate first strand cDNA synthesis, 20 ~.g of BL3 or HL60 ( for human cDNA library construction) poly (A) RNA was reverse transcribed into cDNA by superscript II

reverse transcriptase (GibcoBRL) with oligo dT and random hexamer as primer following BRL's instructions. About 30% of poly(A) RNA was converted into cDNA. The synthesized cDNA:RNA hybrid was size-fractionated through Sepharose CL-4H column (Pharmacia) to remove small cDNA.

Three ~,g of first strand cDNA:RNA hybrid was used for second strand cDNA synthesis . RNA strand was replaced with DNA strand by using RNAse H, DNA polymerase I, E.

toll DNA ligase and T4 DNA polymerase (H1~). EcoRI

recognition sites in dsDNA were methylated by EcoRI

methylase (Promega) to prevent digestion by EcoRI to be carried out in a later step. Three different EcoRI

linkers (8mer, lOmer, and l2mer) were used for ligation with ds cDNA in 100:1 molar ratio of linker:cDNA to create three different reading frames for translation of any cDNA in the library. After ligation, EcoRI digestion was performed to generate EcoRI cohesive ends in each cDNA molecule. Excess EcoRI linkers were removed by size-fractionation through a Sepharose CL-4B column.

Next, to ligate with phage vector and packaging into phage particles, the (doubled-stranded) ds cDNA with EcoRI sites were ligated with ~gtll/EcoRI vector (Stratagene) and packaged into phage particles using phage package extracts (Stratagene) following the vendor's instructions. The size of cDNA library was determined by titering the packaging mixture, i.e., infection of bacteria Y1088 with diluted packaging mixture. A total of 2 ~,g of Agtll/EcoRI vector and 0.45 ~g of ds cDNA were used for ligation. For the HL50 sample, five packaging extracts were used. The size of the ~gtll-HL60 cDNA library is 1.35x106 pfu. For the BL3 library construction, four packaging extracts were used.

The size of the ~gtll-BL3 cDNA library is 1.5x10' pfu.

The libraries were amplified once by infection of bacteria Y1088. To determine the average size of cDNAs in the library, 18 phage clones were randomly picked up for analysis. Phage DNA was extracted and digested with EcoRI to release the cDNA inserts. The average size of cDNAs was obtained by dividing the total size of EcoRI

fragments from all 18 phage DNA samples with 18, giving a value of l.4kb.

For screening the phage cDNA library, appropriate amounts of SM1 monoclonal antibody (MAb) first antibody to be used for gene screening were predetermined by incubating serially diluted antibody supernatant, as well as supernatant of YB2/0 myeloma line (negative control), with lysates of HL3 cells in parallel with that of E.

coli as a control. By an immunodiffusion method well-known in the art, SM1 monoclonal antibody has been shown to be the IgM form. Likewise, optimal amount of alkaline phosphatase conjugated second antibody was also predetermined. Alkaline phosphatase conjugated anti-rat light chains (K and ~) monoclonal antibody from Sigma and alkaline phosphatase conjugated anti-rat IgM (~-chain specific) antibody from Rockland were tested for specific interaction with SMl MAb. Rat IgM (from Rockland) and E.

coli phage lysate (from Stratagene) were used as negative controls. Five fold serial dilutions of each protein were made in blocking solution from 10 ~cg/ml of starting concentration to 2 ~,g/ml, 0.4 ~cg/ml and 0.08 ~,g/ml. One ~cl of each solution was spotted onto a nylon membrane.

Four identical membranes were made and each of them was used for blotting with different antibodies in different dilution. The membranes were shaken in blocking solution for 1 hour at room temperature. Each membrane was WO 98/50429 PC"T/U598/08829 incubated in different secondary antibody solutions, i.e., 1:2000 and 1:10,000 dilution of anti (rc and ~) and anti ~.. Anti IgM a chain specific antibodies gave more specificity, stronger signal and lower background to 12A6 IgM antibody than anti rc and ~ light chain monoclonal antibodies using the same dilution (1: 10,000). So, anti-IgM ~ chain specific antibodies with 1:10,000 dilution was used in antibody screening experiments.

The cDNA library then was screened with anti-SM1 antibody under optimized conditions according to manufacturer's instruction (Stratagene, La Jolla, CA).

A loop of Y1090R bacteria grown in LH plate with 50 ~Cg/ml of ampicillin was inoculated into 15 ml of LB

supplemented with 0.2% maltose and 10 mM MgS04. The culture was incubated at 37C with shaking until the O.D.~ reaches 0.5-1Ø The bacteria were pelleted and resuspended in lOmM MgS04 to 0.5 O.D.~/ml. A 0.6 ml aliquot of bacteria was mixed with ~gtll-BL3 library phage stock containing 50,000 pfu and incubated at 37C

for 15 minutes . Eight ml of top agar ( 0 . 7% agaro9e in NZCYM) was added to the mixture and plated onto a 150 mm NZCYM plate. Twenty such plates were prepared and were incubated at 42C for 3.5 to 4 hours until clear plaques grew up. Dry nylon membranes (from MSI) pretreated with 10 mM IPTG were applied onto the plates and the plates were incubated at 37C for 3.5 hours to transfer the plaques onto the membranes. The membranes were removed from the plates and washed in THST (20 mM Tris.Cl pH

7.5/150 mM NaCl/0.05% Tween 20) 4 times for 15 minutes per wash. They were further blocked in blocking solution (1% BSA in THS t20 mM Tris.Cl pH 7.5/150 mM NaCl)) for at least 1 hour to prevent nonspecific signals. After that, 10-fold diluted SMl monoclonal antibody culture supernatant was added into blocking solution at 8 ml/membrane and incubated with agitation at room temperature f or 3 hours . Next , the membranes were washed 5 times in TBST for 5 minutes per wash and incubated in fresh blocking solution containing secondary antibody conjugated with alkaline phosphatase (Rockland, anti-Rat IgM(~)-AP, 1:10,000 dilution) at room temperature for 3 hours with gentle shaking. Finally, the membranes were washed in TBST and incubated in color development solution !1:50 dilution of NHT/BCIP stock solution from HMH with O.1M Tris.Cl pH 9.5/50 mM MgCl~/O.1M NaCl) for 5-10 minutes in the dark, and the results were recorded.

From about 10 million plaques screened, ten strongly positive clones were identified. Mapping and hybridization studies showed that six clones were identical , two did not contain insert DNA, and two others were not analyzed thoroughly. DNA from two out of the six strong positive clones were sequenced.

Sequence analysis of a positive clone expressing SM1 revealed the partial nucleotide sequence described in Figure 1. A search in GenBank, using the BLAST network service of National Center for Biotechnology Information (NCBI) and from database of non-redundant GenBank+EMBL+DDHJ+PDB sequences, indicates that there is-no homology between SM1 DNA sequence with any other sequence less than 100 base pairs long. After conversion into amino acid sequence, one reading frame translates to a protein having less than 30s sequence homology with other known proteins such as the yeast glucoamylase precursor (Accession #P08640), glycoprotein X precursor (Acc #P28968), yeast alpha-agglutinin attachment subunit precursor (Acc #P32323), spore coat protein sp96 (Acc #1103869), Bovine herpesvirus gp80 (Acc #z84818, e300478), integumentary mucin c.1 (Q05049), or microfilarial sheath protein (Acc # 1163086, U43510).

SM1 Gene Expression Expression of the mouse SM1 gene was examined at both the RNA and protein level. Northern blot analysis was performed on samples from various mouse organs using a northern blot filter purchased from Clontech (Palo Alto, CA). A l.Skb EcoRl fragment of SM1 or y-actin DNA

was used as the probe. aybridization and subseauent washing was done according to the standard procedure specified by manufacturer's recommendation. After three days exposure to the X-ray film, a specific 7.5kb fragment hybridized with the SM1 probe was observed in most tissues. The intensity of the band was highest in testis and lowest in spleen and lung, after normalization with that of the y-actin probe. Thus SM1 gene appears to be expressed ubiquitously at the RNA level. Similar results were obtained on RNA extracted from various cell lines including BL3, EL4, WEHI-3 and 70Z/3, revealing the presence of the same 7.5kb SM1 RNA band (data not shown).

SM1 protein was characterized by immunoprecipitation according to the following procedure. Twenty million BL3 ~5 cells were harvested and washed twice with P2 buffer (PBS

plus 2% FCS). The cell pellet was resuspended with 0.5 ml P2 buffer and incubated with 10 ~g IgG for two hours at 4C. The cells were washed twice with P2 and lysed with the same lysis buffer as described for western blot.

20 The cell lysates were placed on ice for 30 minutes, spun and the supernatants transferred into the tubes containing 40 ~l Protein A-agarose suspension (50% volume swollen agarose, BMH). They were incubated for a further two hours at 4C. Complexes of antigen-antibody-protein 25 A-agarose were collected and washed three times with lysis buffer. The pellets were resuspended with 40 ~,1 of 2X sample buffer, boiled for 3 minutes and spun for 2 minutes at room temperature. Supernatants were collected and separated by 7% SDS-PAGE.

30 Immunoprecipitation of 35S-methionine labeled cells with SM1 antibody indicated that only BL3 cells expressed SMl protein on the cell surface, whereas other cell lines expressing the SMl RNA did not. See Figure 2.

As indicated above, the present invention in one 35 aspect relates to SM1 protein, substantially purified from other proteins that has a molecular weight of about 230 kDa, as measured by irtununoprecipitation and SDS-PAGE, and chat .s present on the surface of human and mouse hematopoietic stem cells and primitive progenitor cells, but absent from those of other cells including rr~DC-P1 myeloid progenitor cells, EL4 T-cells, WEHI-3 S myelomonocytic cells, and 70Z/3 pre-B lymphoid cells, or from differentiated hematopoietic cells of human cord blood or mouse bone marrow. The invention also includes peptide fragments of SM1. Such peptide fragments could represent research and diagnostic tools in the study of zematopoietic stem cell development. In addition, pharmaceutical compositions comprising isolated and purified peptide fragments of SM1 may represent effective therapeutics against various diseases such as acquired i~nunodeficiency syndrome (AIDS).

A search in GeneBank using SM1 DNA sequence indicates that it has weak sequence homology to one encoding a receptor molecule. Recently, chemokine/cytokine receptor molecules have been implicated in the process of human immunodeficiency virus (HIV) infection, and HIV viral entry is thought to require more than one receptor molecule. Cocchi et al., Scieace 270:1811 (1995); Paxton et al., Nature Med. 2:412 (1996); Dragic et al., Nature 381:661 (1996); Simmons et a1. , Science 276:276 (1997) . Blockage of virus entry can be achieved as a result of cytokines or chemokines binding to their corresponding receptors. SM1 likewise may be a novel receptor, such that binding by its ligand would block HIV viral entry and, hence, render target cells resistant to HIV infection.

The invention relates not only to fragments of naturally-occurring SM1 but also to SM1 mutants and chemically synthesized derivatives of SM1. For example, changes in the amino acid sequence of SM1 are contemplated in the present invention. SM1 can be altered by changing the DNA encoding the protein.

Preferably, only conservative amino acid alterations are undertaken, using amino acids that have the same or similar properties. Illustrative amino acid substitu~ions include the changes of: alanine to serine;

arginine to lysine; asparagine to glutamine or histidine;

aspartate to Qlutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline;

ristidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine;

lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, y~ leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.

Additionally, other variants and fragments of SM1 can be used in the present invention. Variants include analogs, homologs, derivatives, muteins and mimetics of SM1. Fragments of the SM1 refer to portions of the amino acid sequence of SM1. The variants and fragments can be generated directly from SM1 itself by chemical modification, by proteolytic enzyme digestion, or by combinations thereof. Additionally, genetic engineering techniques, as well as methods of synthesizing polypeptides directly from amino acid residues, can be employed.

Non-peptide compounds that mimic the binding and function of SM1 ("mimetics") can be produced by the approach outlined in Saragovi et al. , Science 253 : 792-95 (1991). Mimetics are molecules which mimic elements of protein secondary structure. See, for example, Johnson et al.,"Peptide Turn Mimetics," in BIOTECHNOLOGY AND

PHARMACY, Pezzuto et al., Eds. (Chapman and Hall, New York 1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions. For the purposes of the present invention, appropriate mimetics can be considered to be the equivalent of SM1 itself.

Variants and fragments also can be created by recombinant techniques employing genomic or cDNA cloning methods. Site-specific and region-directed mutagenesis techniques can be employed. See CURRENT PROTOCOLS IN

MOLECULAR BIOLOGY vol. l, ch. 8 (Ausubel et a1. eds., J.

Wiley & Sons 1989 & Supp. 1990-93); PROTEIN ENGINEERING

(Oxender & Fox eds., A. Liss, Inc. 1987). In addition, linker-scanning and PCR-mediated techniques can be employed for mutagenesis. See PCR TECHNOLOGY (Erlich ed., Stockton Press 1989); CURRENT PROTOCOLS IN MOLECULAR

BIOLOGY, vols. 1 & 2, supra. Protein sequencing, structure and modeling approaches for use with any of the above techniques are disclosed in PROTEIN ENGINEERING, loc. cit., and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vols. 1 & 2, supra.

Hematopoietic stem cell enrichment using anti-SM1 antibody CFU-S-forming cells are multipotent hematopoietic progenitors capable of reconstituting lethally-irradiated recipient mice short-term. CFU-S spleen-focus assays were performed as described in Wong et a1. ( 1994 ) , supra, using donor adult bone marrow cells. Donor mice were inbred male C57BL/6J (Jackson Laboratory, Bar Harbor, ME), and recipient mice were female of the same strain.

To prepare labeled bone marrow cells, male mice were sacrificed by cervical dislocation, and bone marrow cells were as we described previously (along et. al. (1994), supra). Mononuclear cells (MNC) were separated by overlaying whole bone marrow (HM) cell population on lymphocyte-separation medium (LSM) at a ratio of 5:3 by volume (cells . LSM) and spun at 1,600 rpm and 25C for 20 minutes. After washing twice with P5 (phosphate buffer saline (PBS) supplemented with 5% FCS and 0.02%

sodium azide), the cells were mixed with FITC-conjugated antibody (Ab), at a concentration of 10x106 cells per 10 ~g Ab/ml, and incubated on ice for 30 minutes.
Ab-labeled cells were washed 3 times and resuspended in P5 at a concentration of 5x106 cells per ml for FRCS

c7 _ analysis and cell sorting. initial analysis suggested that 1-5% of bone marrow cells may stain positive with SM1 hybridoma cell supernatant using indirect immunofluorescent assay. Because primitive stem cells or progenitor cells comprise a small fraction in the bone marrow, 0.1% of BM cells brightly stained with SM1 were sorted. These sorted male cells were used as a source of donor cells.
For recipient female mice, each of them was irradiated with a dose of 9.5 Gy prior to engraftment with SM1 sorted cells. Irradiation was done by using a cesium source Mark 1 (model 30-1) irradiator (JL Shepherd & Associates, San Fernando, CA). Varying numbers of sorted SM1 cells suspended in 0.5m1 of R2 medium were subsequently engrafted intravenously into the irradiated female recipients. Twelve days later, CFU-S spleen foci were individually dissected from either the recipient mice of 100 to 1,000 SM1 sorted male donor cells, of control unsorted male bone marrow cells or female accessory cells. DNA was then extracted from each of these foci.
To extract DNA, each dissected -CFU-S focus was placed into an Eppendorf tube containing 0.5m1 of PBS, and single cell suspension was prepared by repeated pipetting. The cells were washed once with PHS and lysed in DNA extraction buffer. This was followed by treatment with 100 ~,g/ml RNAse at 37°C for one hour, and 100 ~cg/ml proteinase K at 56°C for 3 hours. DNA then was extracted twice with phenol/chloroform and precipitated with 2M
ammonium acetate and 2X volume of absolute ethanol. The DNA was dissolved thereafter in 0.4 ml of TE buffer and the concentration of DNA was determined. To examine whether the foci originated from donor SM1+ cells, the pY2 probe was used. This probe has been shown to be relatively specific for the Y chromosome in male cells.
Lamar & Palmer, Cell 37:171 (1984). Because the probe was not absolutely specific for male chromosome, a two-step analysis was done: a dot blot analysis was performed on all samples first, followed by southern blot analysis ca samples tested positive on dot blots.

For dot blot analysis, 5 microgram DNA of each sample was mixed with 0.1 volume of 3M NaOH and incubated or ~ 0 -.~.inutes at 65 C to denature DNA and to destroy RNA. __ was then neutralized with 0.1 volume of 2M

ammonium acetate pH 7.0, and blotted onto NYTRAN nylon Filter. Four-fifth of a sample was used for hybridization with pY2 probe and one fifth with a GAPDH

probe. ?ositive samples would then be used for southern blot analysis to confine the presence of Y-specific band using the pY2 DNA fragment as probe. For southern blot analysis, typically 10 ~,g of DNA was digested with BamHl restriction enzyme, and the digested DNA was processed, transferred to nylon filter and hybridized with a random primer-labeled pY2 probe.

DNA of some CFU-S foci from recipients of 100 SM1+

cells hybridized positively with the pY2 male-specific probe (Figure 3). Those that were negative presumably derived from endogenous short-teen CFU-S forming cells.

Each of these CFU-S foci has been shown to contain differentiated erythroid and myeloid cells. To give a positive signal from dot-blot or southern blot analysis, approximately 1.5 million cells are required to give 15 ug of male-specific DNA. These results therefore indicate that some SM1+ donor cells are multi-potential short-term hematopoietic stem cells.

SM1- cells in the mouse bone marrow was estimated to be about 1-5s. To investigate whether hematopoietic stem cells reside in a subset of SM1+ cells, cells that were positive with lineage specific markers, i.e., CD4 (T

helper cells), CD8 (T killer cells), Gr-1 (granulocytes), TER119 ~ezythroid cells), Mac-1 (macrophages) and B220 (pre B cells), were depleted. These Liri cells (for lineage negative) were further divided into SM1T and SM1-cells. FACS analysis was performed on mouse bone marrow WO 98/50429 PC'f/US98/08829 cells by using PE (polyerythrin) conjugated antibodies directed against all the lineage specific markers and FITC-conjugated SM1 antibody. Figure 4 indicates the result of such a two-color analysis.

To detect the presence of primitive stem/progenitor cells, SM1~/Lin~-sorted cells were plated into semi-solid methylcellulose clonogenic culture familiar to one skilled in the art. The details of the assay are described by Han et al., PNAS, 92:11014 (1995), which is incorporated herein by reference. The experiment this time was done by sorting out SM1+/Lin- cells, which comprise of 0.06% of mouse mononuclear cell population (0.3% x 0.2% [% area A] - 0.06%). About 1,000 cells were plated into each dish under the conditions in which either pokeweed-mitogen stimulated spleen cell conditioned medium (SCM) or BL3 conditioned medium (HLCM) was present. Seven and twelve days later, the numbers and types of hematopoietic colonies were recorded.

No colonies could be observed in the absence of conditioned medium, a source of growth factors. Under the condition in which SCM was present, multilineage mixed type colonies consisting of different types of terminally differentiated cells were present at a significant frequency (Table 1). Earlier than the multilineage colonies are the blast colonies, which became more obvious on the twelfth day after initiation of culture. Cells in the blast colonies have been shown to have CFU-S forming capability and therefore some of which are at least at the stage of CFU-S forming cells, i.e., short-term hematopoietic stem cells. Of note is the presence of novel, compact colonies. These colonies are tight aggregates of undifferentiated cells and could be found only when either SCM or BL3CM was present in the culture. Previously, BL3CM was shown to contain a unique stem cell activity but devoid of many known hematopoietic growth factors. Wong et a1. (1994) supra. This stem cell activity of BL3 has also been shown to be present in SCM. Thus, cells in the compact colonies could be even earlier than those present in the blast colonies.
After twelve days of culture, compact colonies were no longer observed in the experimental condition in which only BL3CM was present (Table 1). After 7 days in culture, these colonies were found to be degenerating.
This is also consistent with the observation that the activity is stem cell specific and it only stimulate stem cell self-renewal, but in order that the colonies are to develop further and expand in size, additional growth factors such as those in SCM would be required.
Table 1. Colony formation ability of bone marrow derived SM1T/Lin- cells.
Numbers & Types of colonies per 1,000 cells per dish Day 7 Day 12 Compact Diffuse Mix Blast Compact Mix Blast CFU-C

1 No GF 0 0 0 0 0 0 0 0 2 SCM 6,5,11 7,6,6 0,1,0 5,3,4 4,5,4 3,2,3 4,4,5 3 BL3CM 5,8,4 0 0 0 0 0 0 0 Numbers and types of colonies were recorded on day 7 and day 12 after initiation of culture. Triplicate dishes for each experimental point were prepared.
Experimental points for which no colonies were observed in all dishes were represented with one zero number.
Definition for colony types is as reported by Han et al.
(1995), supra, except for the novel compact colonies.
Compact colonies are those tight-appearing aggregates of undifferentiated cells with an estimated average size of 50-200 cells.

- ~1 -Characterization of human SMl DNA

To isolate the human counterpart of SM1 gene, a gtll cDNA library was constructed using mRNA of HL60 cell line, which is a human myelomonocytic leukemic cell line and which expresses three mRNA specific to the mouse SM1 DNA. Construction cf ~iL60 cDNA library is similar to what was done on the construction of HL3 ~gtll cDNA

library, as already described. A l.5kb EcoRl mouse SM1 fragment was used to screen the HL60 cDNA library.

Several positive clones were obtained. DNA of two clones were sequenced and one region with the sequence as shown in Figure 5 was found to be common to both DNA samples.

A search in the EST library of GenBank indicates that this sequence is homologous to a homo sapiens cDNA (for example, accession number H98251); no known function for this cDNA has ever been reported.

Expression of human SM1 Qene To examine whether the hu-SM1 gene is expressed, northern blot analysis was done on RNA samples from various human organs (Clontech, La Jolla) and human cell lines. While there was the expected single 7.5kb band in BL3 RNA, there were three bands (9.Okb, 7.5kb and 4.Okb) in RNA of HL60 myelomonocytic cell line, while there was only a 4.Okb band in RNA of K562 cells. On Northern blot of RNA from human organs, all three bands were observed in most of the samples, with the 4.0kb band being most dominant, and the 9.7kb and 7.5kb bands being more abundant in testis and ovary, especially after the intensity of the signals was normalized with that of y-actin.

Three species of mRNA in human cells hybridized . positively with the mouse SM1 probe. Among these species, only one may be responsible for cell surface expression of the human SM1 protein. The three species of mRNA may be related by way of differential splicing, accounting for the fact that common RNA sequence is shared among these species of mRNA. Alternatively, these species of mRNA may represent the product of three distinct genes that are members of a single gene family.

The biological significance of the presence of three RNA species in human cells is not known. Whether they are the products of three distinct but related SM1 genes or the result of differential splicing is unclear.

~lotably, CD34 genes transcribe to produce two species of mRNA, and these are the result of differential splicing Suda et al., Blood 79:2288 (1992); Nakamura et al., Expt.

Hematol. 21:236 (1993). In the context of hematopoietic stem cell enrichment, it is possible that not all species of SM1 RNA will result in production of SM1 protein. It 's therefore important to point out that the 9kb and the 7.5kb bands are abundant in testis and ovary, similar to that of the SM1 7.5kb RNA in the mouse testis. This result is also consistent with the finding in CD34 that full-length but not truncated CD34 inhibits hematopoietic cell differentiation. Fackler et al., Blood, 85:3040 (1995) .

Analysis of various human cell lines indicated that HL60 cells expressing all three species of SM1 mRNA, also express the SM1 on their cell surface; whereas K562 cells expressing only the 4kb species did not express the SMl protein on their surface. SM1 protein also is detected weakly in another cell line J45.

Lysates of various cell lines were immunoprecipitated with SM1 antibody and the immunoprecipitates were resolved on SDS-PAGE. BL3 cell lysate was used as a positive control. Five million cells per sample were labeled with 35S-methionine (0.25 mCi) for 1 hour and then immunoprecipitated for 2 hours at 4C with 20 ~.g SM1 antibody. The cells were then washed with PHS and lysed in 0.5 ml of IP buffer (130mM

NaCl, lOmM Tris.Cl pH 7.5, 5mM EDTA, to Triton X-100 and protease inhibitors). The lysate was cleared by centrifugation. Then 4 ~,g of goat anti-rat IgM antibody was added to each sample and the samples were incubated overnight at 4C. The protein-antibody complexes were pulled down by protein Gt agarose, samples boiled and resolved cn 5% SDS-PAGE. The gel was then fixed for 30 minutes, with 25% isopropanol and 10% acetic acid and treated with an enhancing solution (Enlightening, DuPont) for another 30 minutes. After that, the gel was dried and exposed to X-ray film.

Human hematopoietic stem cell enrichment usincr SM1 antibody SM1 monoclonal antibody also can recognize human hematopoietic cells (Figure 6). FRCS analysis was therefore carried out to examine the proportion of cells that express SM1 molecule on their cell surface. To do that 1 million mononuclear cells from human cord blood were first stained with a mixture of antibodies, which contain rat-anti-CD38, rat-anti-glycophorin A and/or anti-CD33 and anti-HLA-DR, together with PE conjugated anti-rat antibodies. These antibodies detect lineage specific antigens and the cells bearing these antigens are called Lin+ cells. After two washes, these cells were then incubated with 100 ~cl of anti-SM1 hybridoma supernatant for 30 minutes on ice. The cells were then washed again and further stained with FITC-conjugated anti-rat IgM secondary antibodies. FAGS analysis were gated at lymphoid population based on right-angle and forward scatter, and then analyzed based on fluorescence intensity. Area A on the left panel shows the distribution of lymphocytes and small cells, within this population hematopoietic stem cells are known to reside.

Reanalyzing and re-plotting area A, as shown on the right panel of Figure 6, shows that SM1+Liri cells constitute about 0.3% of the whole cord blood mononuclear blood sample (shown as 0.4% in compartment 4). Using SM1 antibody alone, 1% of human cord blood mononuclear cells are found to carry the SM1 antigen. Hematopoietic stem cells have been found to be present in human cord blood at a very high frequency. Xiao et al., Blood 20:455 (1994). By contrast, the CD34 antigen, which has also been used for hematopoietic stem cell enrichment, Haylock et al., Blood 80:1405 (1992), has been shown to occur on about 20 of cord blood cells, Broxmeyer et al., PNAS
86:3828 (1989), 2% of bone marrow cells and 0.20 of peripheral blood cells. Bender et al., Blood 77:2591-2596 (1991).
To examine one aspect of hematopoietic stem cell activity, SM1+/Lin--enriched cord blood cells, constituting 0.30 of the total cord blood mononuclear cell population, were examined by the clonogenic assay.
One thousand sorted cells were plated in methylcelluiose culture in the presence or absence of conditioned medium from 5367 cells derived from a patient with a bladder carcinoma; the conditioned medium (CM) is known to contain various hematopoietic growth factors capable of stimulating primitive hematopoietic stem/progenitor cell growth. Broxmeyer et al., supra. After 10 days of incubation in the presence 10°s 5367CM, blast colonies containing cells dispersed diffusely could be observed (Table 2). In the absence of 5367CM, no colonies were observed. These data indicate that SM1+/Liri-enriched cell population contains primitive hematopoietic stem/progenitor cells.
Table 2. Blast colony formation by human cord blood SM1+/Liri cells Number of blast coloaies per 1,000 cells per dish 1. Without 5367CM 0 2. With 5367CM 3,2,3 In one embodiment of the present invention, anti-SM1 antibody is used to prepare a composition enriched for hematopoietic stem cells. This is achieved by providing antibody that binds SM1, immobilizing anti-SM1 antibody on a support platform such that the antibody retains its SM1-binding capability, then bringing a mixed population of cells into contact with the antibody, where the mixed population contains hematopoietic stem cells, such that the stem cells adhere to the support platform, and removing nonadherent cells, so that a population enriched for hematopoietic stem cells remains adhered to the support platform. 3y "support platform" is meant any solid support such as beads, hollow fiber membranes, resins, plastic petri dishes, or an antibody against the anti-SM1 antibody.

The antibodies may be conjugated with markers such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support , fluorochromes , which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type.

Any technique may be employed which is not unduly detrimental to the viability of the remaining cells.

As has been the case with anti-CD34 antibody and a biotinylated second antibody put through an avidin column to remove breast cancer cells in human transplants Hensinger et al., J. Clin. Aphersis 5:74-76 (1990);

Herenson et al., Blood 76:509-515 (1986). Preferred methods of separation include column chromatography, fluorescence-activated cell sorting, magnetic bead separation, and direct immune adherence.

In another embodiment, the invention relates to a kit for detecting a hematopoietic factor that binds to SM1. Hy "hematopoietic factor" is meant any protein associated with hematopoiesis. This kit comprises the antibody of the present invention, and also can comprise a detectable label and a set of written instructions for using such a kit. Such a kit may comprise a receptacle being compartmentalized to receive one or more containers such as vials, tubes and the like, such containers holding separate elements of the invention.

In another embodiment, SM1 is used in a method of detecting in a sample a hematopoietic factor that binds SM1. Such methods may be used to detect and evaluate WO 98!50429 PCT/US98/08829 .actors associated with the regeneration, differentiation, and maturation of hematopoietic cells.

SM1, and SM1+ cells, may be used in assays to determine .he activity of media, such as conditioned media, and to avaluate fluids for cell growth activity, involvement Faith dedication of particular lineages, or the like.

'.'his in vitro assay involves contacting a sample suspected of containing a hematopoietic factor that binds SM1 with detectably labeled-SM1. The hematopoietic .actor is then detected. By ~~sample~~ is meant any cell culture medium or any body fluid or tissue, including blood, urine, saliva, spinal fluid, semen, peritoneal 'luid, and tissue from any part of the body. Such assays :nay involve binding SM1 to a solid surface. Many methods for immobilizing biomolecules on solid surfaces are known in the art . For instance, the solid surface may be a membrane (e.g., nitrocellulose), a microtiter dish or a bead. The bound molecule may be covalently or noncovalently attached through unspecific bonding. The manner of linking a wide variety of compounds to various surfaces is well-known and well-documented in the literature. See, for example, Chibata, Immunological Enzymes, Halsted Press (1978), and Cuatvecasos, J. Eiol.

Chem. 245:3059 (1970), the respective contents of which are incorporated herein by reference.

In the assay of the present invention for detecting hematopoietic factors that bind SM1, SM1 is labeled by methods well-known in the art. A common method involves the use of radioisotopes such as 3H, 'ZSI , 3sS , 'C or 3zp .

Detection is accomplished by autoradiography.

Non-radioactive labels include the covalent binding of biotin to the compound of the present invention. Biotin is then bound to an anti-ligand such as streptavidin, which is either inherently labeled or bound to a signal system, such as a detectable enzyme, a fluorescent or chemiluminescent compound.

In another embodiment, SM1T cells may be employed to facilitate better characterization of molecular mechanisms and cellular interactions involved in the regulation of SM1 self-renewal and commitment to differentiation of populations derived therefrom. Such mechanisms may, for example, involve any molecule or factor, hematopoietic or not, that is associated with or interferes in SM1 mediated signal-transduction.

Hematopoietic cells purified according to the present invention can also be used in a method of gene therapy. Such methods may comprise gene constructs, which include those mediated by viruses (e. g., retrovirus, adenovirus, adeno-associated virus, Epstein-Barr virus, hepatitis virus, lentivirus), and non-virally mediated methods such as gene transfer into the purified cells. Methods of retrovirally-mediated gene transfer are known in art Bodine et al., PNAS, 86:8897-8901 (1989), but heretofore it has not been possible to use such homogenous population of cells having SM1 as the cells transfected. Such transfected cells can then be used for therapeutic applications.

Treatment of genetic diseases by genetic modification of SM1 cells to correct the genetic defect.

For example, diseases such as B-thalassemia, sickle cell anemia, adenosine deaminase deficiency, etc., may be corrected by the introduction of a wild-type gene into the SM1 cells . Other indications of gene therapy include introduction of viral or bacterial resistance genes, antisense sequence or ribozyme to prevent the proliferation of the pathogen in the SM1 hematopoietic cells. Alternatively, diseases associated with an overproduction of a particular secreted product such as hormone, enzyme, or the like, the SM1 hematopoietic cells may also be inserted with a ribozyme, antisense, or other inhibiting factor to inhibit the particular disease.

Without =urther elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent.

Claims (13)

WHAT IS CLAIMED IS:

1. A protein, substantially purified from other proteins, that has a molecular weight about 230 kDa, as measured by immunoprecipitation and SDS-PAGE, that is present on the surface of human or mouse hematopoietic stem cells and primitive progenitor cells but that is absent from the surface of cells selected from the group consisting of FDC-P1 myeloid progenitor cells, EL4 T-cells, WEHI-3 myelomonocytic cells, and 70Z/3 pre-B
lymphoid cells, differentiated hematopoietic cells of human cord blood, and differentiated hematopoietic cells of mouse bone marrow.

2. An antibody against the protein of claim 1.

3. An antibody of claim 2, which is a monoclonal antibody.

4. A method for preparing a composition enriched for hematopoietic stem cells, comprising the steps of (a) providing antibody that binds SM1, (b) immobilizing said antibody on a support platform such that said antibody retains its SM1-binding capability, then (c) bringing a mixed population of cells into contact with said antibody, wherein said mixed population contains hematopoietic stem cells, such that said stem cells adhere to said support platform, and (d) removing nonadherent cells, whereby a population enriched for hematopoietic stem cells remains adhered to said support platform.

5. A kit for preparing a composition enriched for hematopoietic stem cells, comprising an antibody against the protein of claim 1.

6. The kit of claim 6, further comprising written instructions for using said kit.

7. A method for detecting in a sample a hematopoietic factor that binds the protein of claim 1, comprising (a) contacting a sample suspected of containing said growth factor with the protein of claim 1, wherein said protein is detectably labeled, and b) detecting the binding of said growth factor with said detectably labeled protein.

8. A kit for detecting in a sample a hematopoietic factor, comprising the protein of claim 1.

9. The kit of claim 8, further comprising a detectable label selected from the group consisting of a fluorescent, a radioactive and an enzymatic label.

10. The kit of claim 8, further comprising written instructions for using said kit.

11. An isolated DNA molecule that encodes protein corresponding to the protein of claim 1.

12. An isolated DNA molecule of claim 11, comprising the nucleotide sequence:
GGAATTCCGN CAGCAAGTTC TTATTCTGCC TAAGAATTTT GTGATTCAGC
ACAAAGAGGG GAAAGCAGTT GAAAAAGAGA TAGCAGCACC TCAGCAGAAA
GGCCCAGAGC ATTGCTCACC TGGCCCACAG ACAAGCGCTA CGTGTTCCTT
AGTGTCTGTT CCTGTCACCT CTGTGTCTAC CCAACTGCCT AATACAGTTC
TCAGTAAGAC AAGTACACCT TCATCAAATG TGAGTGCTAG ATCACAGCCT
TTGTCTCCTG TAGCCTCTGT AAGTAATGCA TTAACATCAC CAGTTAAGAC
TAGCCAAAGT GAAGCAGGAA AAGTCAAGAG TACCGCTTCA TCCACCACAC
TCCCCCAGCC TCACACTTCA CCTACCATTT CATCAACAGT TCAGCCTCTC
TTGCCAGCAA CAACACTAAA TGAATCTACA GATCCTGGCA GTTCCATCCC
CTGTTTTTCA CAGCAAACTG TTGATTCTTC TGAGGCAAAG CAAGAACTAA
AAACTGTATG TATACGAGAT TCACAGTCAA TTCTTGTTAG GACTCCAGGT

GGGAACACTG GAGTTGTAAA AGTACAAACT AATCCGGAAC AAAATTCACC
CAACAGTTTA TCTTCAAGTT CTGTTTTCAC CTTTACACCT CAATTTCAGG

CATTTCTTGT GCCAAAATCA ACATCATGCT CTGCTTCCTC ACAAGTAGCC
GGAGTGACTA CTACATCTAG TCTACCATCT TTCAGCCAAG CAATCTACGT
NTGTGTNGCT TCATCCACCC ATGGGAAAAA TCTCAAATCT ACACAAGGCC
AAACCTTGAG CAGTGGTATG TAGGCCCCAT GATAGAAAAA ACGTCATACA
TGCCCTCTTC ACCCTTGAAG CCTTCTGTTT CTTCCAGCTC ACTGCTACCA
TCAACAACAA ATAGTTCAGT GAGTGTAATT AGCATATCAA CAGGAAATNN

NGGGCAAACC AATACAAATG TTATTCATAC ATCAACTAAA CCACAACAAG
TAGATTGTAT CACNAAAAGT TACCCAGTTA CAAGATCAGA AGCAACAACA
GCAGTAAATG GTGATGTGCT CGGTGAGACT CCAGGTCAGA AACTGATGCT
GGTGTCAGCT CCATCTGGTC TCCCTTCTGG CAGTGTACCT TCAGTTAACA
CGGCACCAGA ACCGACATCT GCAGGTGTGT CTACCCAGAA GGTAGTTTTT
ATTAATGCTC CAGTTCCTGG TGGCGCTTCA TCCTCAGCTA TTGTTGCAGA

ATCATTAAGA CAGTCACTTC CTTCTCCCAC AAATACTGTA TTACTAGTGT
GCTTGTAGTA GTTAACTCCA CCATCTTTGT AAGCTAATGA AATTGTGAGT
CACCCATTTA TATCTTAATT TTTAATCATG TCAGTTCTTG AATGGGTATC
TCCTTAGCCT GCTGATTTCT TTTTCTTTCT AAAGAAAGTG GGTGGAGAAA
TTAATTTAGA CGTTTGTTTG CAATAAAAAG AATTC

13. An isolated DNA molecule of claim 11, comprising the nucleotide sequence:
GAATTCTTTT TATTGCAAAC AAACGTCTAA ATTAATTTCT CCACCCACTT
TCTTTAGAAA GAAAAAGAAA TCAGCAGGCT AAGGAGATAC CCATTCAAGA
ACTGACATGA TTAAAAATTA AGATATAAAT NGGTGACTCA CAATTTCATT
AGCTTACAAA GATGGTGGAG TTAACTACTA CAAGCACACT AGTTATACAG
TATTTTGTGG GAGAAGGGCA TACAGACATG GCTAACTTCA TATAGATCCC
ATTAGACAAC TGGATTTACA ACAAGTTTTT TTAATAAGAA ATGGGCAAAG
CAGCTTTCTT TTCAGAATCA AAATGCAGAA CAAATGGAAA AATTATGGTA
TTAACCTTCA CAAGTTTGAG CCTCCACAAA TAATGCAACC AAGTTTTACA
TTTTTAACAG CCCTTCTACA TACACTCCAT CTTCTCTATC TTAGTTCCAA
GTTTTAGTTT TCAATCCCAA TTATACCAAT TCCATTGTTA TTTTAAGAAA
AAACCTTCCC AGTTATTGTC AGAAACTATG ATTTAGCTTA CCCCCTCCAC
TACNNAGCAA ACTACAGAGA GGATGGAGTG TAATATGAGC AGTACAGAGT

CTTAATGCAA TTCATGAGGA CCACTTAGTC CTTACATGAA TCTGGTTGCT
AACATTTCTA TTATATTGTG ACAATGACTC CCGACTGTTA TTCTCTGTGA
GAAATGGGGG GAGTAAATTC TTAATAAAAG ACACCAGGTA CAAAGCAACA

TTTTACTTCT GTTGTGATAA AAAAAAAAAA AGGTCACATT TTCAGATAAA
ATGTGGAACC CTGAAATCTG ACACATTCTC TTATCGTGCC ACCAATGCTG
AGGTTCTCTT ACGATTCACT TTTAAACTGC AATTAAAAAT GTACAAAAAA
GAAAAGAAAA AAANTCAACC CACAAAGCTT CTAAAAAAGG AACCCGCAGG
CACTTCCTCT TGTGGAATGT TTAAAAAGTT AGCCTACTAA AGAAAACAGT
CGACTTCTTG TGAAGGTTTT GGAGAAATAT GTATCAGTTC GTTTTATTTG
GGTATTCAAT AATATCCTTG GTGATAATGC TGACTCCATG GCTTCTGACC
CCAGAATTGA CCCTGCTGCC ACTGGTTGTA GCCCTGAGAT TGATTTTTGT
AGCCACGATT GTTTCCTCGT CCTCTGAAGT TCTGGTTGTA GTTCCCTCTG
TTGGGCATTC CACCTCTGTT GTAGTTCCCT CTGTTTGAGT AACTACCACG
GCCAGGAAAA ACAGGGGCAC GAGGGTATGG ATAGCCGATT CCACCACTTC
CTCCACCGCC ACCACCTCTC TGTGGCATGT TGCCCTCCTA TTATATCCGC
CACGATTCCC AGGGGCTCCT CCTCTGAAAT TTCCACCACG CATATTGAAT
CCTCCACGTC TCTATGGCCA CCACCTCTGT TAAACTGGTT CTTGCCACTC
TTATTTTTAT TGCTTTTCTT TGAGCCAGTG TTCTGTTTCT TTTCTGGTGG
AAGAGCCTTT TTGCTTTCTT CCTTATATTG CTCCAAGAGT TTTTGGGCTT
CTTCCTTCTG AAGGGCAACA TAGGTTATTT CATCAAAGCA CTCAGCTACC
TCTGGGAGGG TAAAGTTTCC TTTCATTT