Note: Descriptions are shown in the official language in which they were submitted.
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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.,
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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
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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
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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
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(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.
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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
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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
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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
2 5 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 AAAGAAAGTG GGTGGAGAAA
TTTTCTTTCT
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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
25 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 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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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,
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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
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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.
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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.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.
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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.
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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.
