GENES THAT REGULATE HEMATOPOIETIC BLOOD FORMING STEM CELLS AND USES THEREOF

GENES REGULANT LES CELLULES SOUCHE HEMATOPOIETIQUES ET UTILISATION DE CES GENES

English Abstract

The present invention provides an isolated nucleic acid derived from an
isolated hematopoietic stem cell. The present invention additionally provides
an isolated hematopoietic stem cell specific protein or a portion thereof
encoded by the provided nucleic acid. In addition, the present invention
provides a method for generating a stem cell/progenitor cell from a primitive
hematopoietic cell. Also, the present invention further provides a method for
identifying the presence in a sample of a compound that modulates
hematopoietic stem cell activity. The present invention additionally provides
a molecularly defined primitive hematopoietic stem cell. Finally, the present
invention provides a method of ex vivo expansion of hematopoietic stem cells.

French Abstract

L'invention concerne un acide nucléique isolé dérivé d'une cellule souche hématopoïétique isolée. Elle concerne également une protéine spécifique de cellule souche hématopoïétique ou une portion de cette protéine codée par l'acide nucléique décrit. L'invention concerne en outre un procédé permettant de produire une cellule souche/cellule précurseur d'une cellule hématopoïétique primitive, ainsi qu'un procédé permettant d'identifier la présence d'un composant modulant l'activité des cellules souches hématopoïétiques dans un échantillon. De plus, L'invention concerne une cellule souche hématopoïétique primitive dont la composition moléculaire a été définie. Enfin L'invention concerne un procédé de culture ex vivo de cellules souches hématopoïétiques.

Claims

94
WHAT IS CLAIMED IS:
1. An isolated nucleic acid derived from an isolated hematopoietic stem cell,
the isolated
nucleic acid comprising the following characteristics:
- specifically expressed in the hematopoietic stem cell; and
- encoding a hematopoietic stem cell - specific protein.
2. An isolated nucleic acid of claim 1, the isolated nucleic acid further
comprising the
following characteristic:
- capable of hybridizing under standard conditions with a sequence selected
from
the group consisting of SEQ.ID.No.: 1, SEQ.ID.No.: 2, SEQ.ID.No.:3,
SEQ.ID.No.: 4, SEQ.ID.No.:5, SEQ.ID.No.:6, SEQ.ID.No.:7, SEQ.ID.No.: 8,
SEQ.ID.No.: 9, SEQ.ID.No.:10, SEQ.ID.No.:11, SEQ.ID.No.:12, SEQ.ID.No.:
13, SEQ.ID.No.: 14, SEQ.ID.No.:15, SEQ.ID.No.:16, SEQ.ID.No.: 17

SEQ.ID.No.:18, SEQ.ID.No.:19, SEQ.ID.No.: 20SEQ.ID.No.:21, SEQ.ID.No.:
SEQ.ID.No.:22, SEQ.ID.No.:23, SEQ.ID.No.: 24, SEQ.ID.No.:25, SEQ.ID.No.: 26

SEQ.ID.No.:27,SEQ.ID.No.:28, SEQ.ID.No.:29,SEQ.ID.No.:30 SEQ.ID.No.:
31, SEQ.ID.No.: 32. SEQ.ID.No.:33, SEQ.ID.No.:34, SEQ.ID.No.: 35
SEQ.ID.No.: 36, SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.:
40, SEQ.ID.No.: 41, SEQ.ID.No.:42. and SEQ.ID.No.: 43, SEQ.ID.No.: 45,
SEQ.ID.No.: 47, SEQ.ID.No.: 49, SEQ.ID.No.:: 53, SEQ.ID.No.:
55, SEQ.ID.No.: 57, SEQ.ID.No.: 59, SEQ.ID.No.: 61, SEQ.ID.No.: 63,
SEQ.ID.No.: 65, SEQ.ID.No.: 67, SEQ.ID.No.: 72 or a portion thereof.



3. The isolated nucleic acid of claim 1, the isolated nucleic acid further
comprising the
characteristic of encoding a protein capable of modulating hematopoietic stem
cell
activity.
4. The isolated nucleic acid of claim 3, wherein the activity is selected from
the group
consisting of hematopoietic stem cell differentiation and hematopoietic stem
cell
replication.

95

5. The isolated nucleic acid of claim 3, wherein the protein is selected from
the group
consisting of a growth factor, a transcription factor, a splicing factor, a
capping factor,
a transport protein, a translation factor, and a replication factor

6. The isolated nucleic acid of claim 1, wherein the nucleic acid comprises
the
nucleotide sequence of SEQ.ID.No.: 72, an analog thereof, or a portion
thereof.

7. The isolated nucleic acid of claim 1, wherein the hematopoietic stem cell
is a
primitive hematopoietic stem cell.

8. The isolated nucleic acid of claim 7, wherein the primitive hematopoietic
stem cell is
selected from the group consisting of an umbilical cord cell, a bone marrow
cell and
a fetal liver cell.

9. The isolated nucleic acid of claim 7, wherein the primitive hematopoietic
stem cell is
selected from the group consisting of a AFT024 cell, a 2012 cell and a 2018
cell.

10. A composition comprising the nucleic acid of claim 1, wherein the nucleic
acid
comprises one selected from the group consisting of SEQ.ID.No.: 1, SEQ.ID.No.:
2,
SEQ.ID.No.: 3, SEQ.ID.No.: 4, SEQ.ID.No.: 5, SEQ.ID.No.: 6, SEQ.ID.No.: 7,
SEQ.ID.No.: 8, SEQ.ID.No.: 9, SEQ.ID.No.: 10, SEQ.ID.No.: 11, SEQ.ID.No.: 12,
SEQ.ID.No.: 13, SEQ.ID.No.: 14, SEQ.ID.No.: 15, SEQ.ID.No.: 16, SEQ.ID.No.:
17,
SEQ.ID.No.: 18, SEQ.ID.No.: 19, SEQ.ID.No.: 20, SEQ.ID.No.: 21, SEQ.ID.No.:
22,
SEQ.ID.No.: 23, SEQ.ID.No.: 24, SEQ.ID.No.: 25, SEQ.ID.No.: 26, SEQ.ID.No.:
27,
SEQ.ID.No.: 28, SEQ.ID.No.: 29, SEQ.ID.No.: 30, SEQ.ID.No.: 31, SEQ.ID.No.:
32,
SEQ.ID.No.: 33, SEQ.ID.No.: 34, SEQ.ID.No.: 35, SEQ.ID.No.: 36, SEQ.ID.No.:
37,
SEQ.ID.No.: 38, SEQ.ID.No.: 39, SEQ.ID.No.: 40, SEQ.ID.No.: 41, SEQ.ID.No.:
42,
SEQ.ID.No.: 43, SEQ.ID.No.: 45, SEQ.ID.No.: 47, SEQ.ID.No.: 49, SEQ.ID.No.:
51,
SEQ.ID.No.: 53, SEQ.ID.No.: 55, SEQ.ID.No.: 57, SEQ.ID.No.: 59, SEQ.ID.No.:
61,
SEQ.ID.No.: 63, SEQ.ID.No.: 65, SEQ.ID.No.: 67, SEQ.ID.No.: 72 or a portion
thereof.

96

11. The nucleic acid of claim 1, wherein the nucleic acid is selected from the
group
consisting of DNA, RNA and cDNA.

12. A vector comprising the nucleic acid of claim 1.

13. The vector of claim 12, wherein the vector comprises viral or plasmid DNA.

14. An expression vector comprising the nucleic acid of claim 1 and a
regulatory element.

15. A host vector system which comprises the expression vector of claim 12 in
a suitable
host.

16. The host vector system of claim 15, wherein the suitable host is selected
from the
group consisting of a bacterial cell, a eukaryotic cell, a mammalian cell and
an insect
cell.

17. An isolated hematopoietic stem cell specific protein or a portion thereof
encoded by
the nucleic acid of claim 1.

18. The isolated hematopoietic stem cell of claim 16, further comprising the
following
characteristic:
- capable of modulating hematopoietic stem cell activity.

19. The isolated hematopoietic stem cell of claim 18, wherein the activity is
selected from
the group consisting of hematopoietic stem cell differentiation and
hematopoietic
stem cell replication.

20. The protein of claim 17, wherein the protein is selected from the group
consisting of a
growth factor, a transcription factor, a splicing factor, a capping factor, a
transport
protein, a translation factor, and a replication factor.

21. The protein of claim 17, the protein having substantially the same amino
acid
sequence as one selected from the group consisting of SEQ.ID.No.: 42,
SEQ.ID.No.:

97

44, SEQ.ID.No.: 46, SEQ.ID.No.: 48, SEQ.ID.No.: 50, SEQ.ID.No.: 52,
SEQ.ID.No.:
54, SEQ.ID.No.: 56, SEQ.ID.No.: 58, SEQ.ID.No.: 60, SEQ.ID.No.: 62,
SEQ.ID.No.:
64, SEQ.ID.No.: 66, SEQ.ID.No.: 68, SEQ.ID.No.: 70, SEQ.ID.No.: 71, and
SEQ.ID.No.: 73.

22. A nucleic acid probe capable of specifically hybridizing with the nucleic
acid of
claim 1 under standard conditions.

23. An antibody capable of specifically binding to the protein of claim 17
without
substantially cross-reacting with a non-stem cell specific protein or homologs
thereof
under conditions permissive to antibody binding.

24. A cell capable of producing the antibody of claim 23.

25. A method for identifying the presence of a primitive hemopoietic stem cell
in a
sample comprising:
(a) contacting the sample with the antibody of claim 23 under conditions
permissive
to the formation of an antibody complex; and
(b) detecting the presence of the complex formed in step (a), the presence of
a
complex formed indicating the presence of a primitive hemopoietic stem cell in
the sample.

26. The method of claim 25, wherein the antibody is labeled with a detectable
marker.

27. The method of claim 26, wherein the detectable marker is selected from the
group
consisting of a radioactive isotope, enzyme, magnetic bead, dye, fluorescent
marker
and biotin.

28. A method for generating a stem cell/progenitor cell from a primitive
hematopoietic
cell in a sample comprising contacting the sample with the protein of claim
17.

98

29. A method for identifying the presence in a sample of a compound that
modulates
hematopoietic stem cell activity comprising:
(a) contacting the hematopoietic stem cell with the sample;
(b) determining the hematopoietic stem cell activity;
(c) comparing the hematopoietic stem cell activity determined in step (b) with
the
activity determined in the absence of the compound an increase or decrease in
hematopoietic stem cell activity indicating the presence in the sample of a
compound that modulates hematopoietic stem cell activity.

30. The method of claim 29, wherein the activity is selected from the group
consisting of
gene expression, replication, differentiation, transplantation, and self
regeneration.

31. A compound identified by the method of claim 29, previously unknown.

32. A method for identifying primitive hematopoietic stem cell-specific
nucleic acids,
comprising:
(a) creating a primitive hematopoietic stem cell cDNA library and a non-
primitive
stem cell immune cell cDNA library; and
(b) subtracting the two libraries, thereby identifying primitive stem cell
specific
nucleic acids.

33. The method of claim 32, step (b) comprising:
(i) contacting the nucleic acids of the stem cell and non-stem cell libraries
with
each other under conditions permissive to hybridization, thereby forming
hybrid complexes;
(ii) separating the hybrid complexes formed in step (b) from the nucleic acids
which did not form complexes;
(iii) isolating the nucleic acids which did not form complexes, thereby
identifying hematopoietic stem cell specific nucleic acids.

34. The method of claim 33, step (ii) further comprising amplification of the
nucleic
acids.

99
35. The method of claim 33, step (iii) further comprising ampliciation of the
nucleic acids
which did not form complexes.

36. The method of claim 33, further comprising displaying the amplified DNA on
a
chromatography gel.

37. The method of claim 32, step (b) comprising differential display of the
two libraries,
thereby identifying primitive stem cell specific nucleic acids.

38. The method of claim 32, step (b) comprising representation difference
analysis of the
two libraries, thereby identifying primitive stem cell specific nucleic acids.

39. The method of claim 32 further comprising cloning the stem cell specific
nucleic acid.

40. The method of claim 32, wherein the stem cell is selected from the group
consisting
of AF024, 2012, and 2018.

41. A nucleic acid identified by the method of claim 32

42. A composition comprising the compound of claim 31 and a carrier.

43. A pharmaceutical composition comprising the compound of claim 31 and a
pharmaceutically acceptable earner.

44. A primitive hematopoietic stem cell specifically expressing one selected
from the
group consisting of: SEQ.ID.No.: 1, SEQ.ID.No.: 2, SEQ.ID.No.: 3, SEQ.ID.No.:
4,
SEQ.ID.No.: 5, SEQ.ID.No.: 6, SEQ.ID.No.: 7, SEQ.ID.No.: 8, SEQ.ID.No.: 9,
SEQ.ID.No.: 10, SEQ.ID.No.: 11, SEQ.ID.No.: 12, SEQ.ID.No.: 13, SEQ.ID.No.:
14,
SEQ.ID.No.: 15, SEQ.ID.No.: 16, SEQ.ID.No.: 17, SEQ.ID.No.: 18, SEQ.ID.No.:
19,
SEQ.ID.No.: 20, SEQ.ID.No.: 21, SEQ.ID.No.: 22, SEQ.ID.No.: 23, SEQ.ID.No.:
24,
SEQ.ID.No.: 25, SEQ.ID.No.: 26, SEQ.ID.No.: 27, SEQ.ID.No.: 28, SEQ.ID.No.:
29,
SEQ.ID.No.: 30, SEQ.ID.No.: 31, SEQ.ID.No.: 32, SEQ.ID.No.: 33, SEQ.ID.No.:
34,
SEQ.ID.No.: 35, SEQ.ID.No.: 3G, SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.:
39,

100

SEQ.ID.No.: 40, SEQ.ID.No.: 41, SEQ.ID.No.: 42, and SEQ.ID.No.: 43,
SEQ.ID.No.: 45, SEQ.ID.No.: 47, SEQ.ID.No.: 49, SEQ.ID.No.: 51, SEQ.ID.No.:
53,
SEQ.ID.No.: 55, SEQ.ID.No.: 57, SEQ.ID.No.: 59, SEQ.ID.No.: 61, SEQ.ID.No.:
63,
SEQ.ID.No.: 65, SEQ.ID.No.: 67, SEQ.ID.No.: 72 or a functional portion
thereof.

45. A primitive hematopoietic stem cell specifically expressing a nucleic acid
identified
by the method of claim 32.

46. A method for treating a condition in a subject comprising administering to
the subject
a therapeutically effective amount of the composition of claim 43.

47. The method of claim 46 wherein the condition is an immune system
condition.

48. The method of claim 46 wherein the condition is leukemia.

49. A method of introducing an exogenous nucleic acid into a hematopoietic
stem cell
comprising contacting the stem cell with the composition of claim 42.

50. A method of ex vivo expansion of hematopoietic stem cells comprising
contacting the
cell with the composition of claim 42.

Description

CA 02340465 2001-02-21
WO 00/11168 1 PCT/US99/19052
GENES THAT REGULATE HEMATOPOIETIC BLOOD FORMING STEM
CELLS AND USES THEREOF
This invention was made with the support of National Institutes of Health
Grant
No. ROl CA45339-09 and American Cancer Society No. DHP-144/01. The United
to States government may have certain rights to this invention.
Throughout this application, various publications are referenced by number.
Full
citations for these publications may be found listed at the end of the
specification and
preceding the Claims. The disclosures of these publications in their
entireties are hereby
incorporated by reference into this application in order to more fully
describe the state of
the art. A Sequence Listing is provided.
FIELD OF~'HE INVENTION
The present invention relates to hematopoietic stem cells and the stem cell
and support
cell genes that support stem cell replication and differentiation.
BACKGROUND OF THE INVENTION
The adult hematopoietic system is organized as a hierarchy of cells with
decreasing self
renewal and multilineage differentiation potential. This is accompanied by
progressively
larger numbers of more mature cells and an increasing tendency to be in active
cell cycle
(Lemischka, LR., 1992; Mornson, S.J., et al. 1995). Collectively, the
properties of this
hierarchical system result in the balanced, lifelong production of at least
eight distinct cell
lineages. A population of stem cells establishes the entire hierarchy;
therefore, in order to
understand the fundamental mechanisms responsible for normal hematopoiesis it
is
ultimately necessary to understand the biology of the stem cells.
3o Most information concerning the biology of stem cells has been obtained
from the
mouse model. In this system, the most critical, characteristic property of the
stem cell
population has been defined; that is, its ability to reconstitute a normal
blood system in a
transplanted host. A number of variations on the basic transplantation assay
have been


CA 02340465 2001-02-21
WO 00/11168 2 PCT/IJS99/19052
described (Harrison, D.E. 1980; Spangrude, G.J., et al. 1995). All of these
systems,
together with the appropriate donor vs. host or clonotypic markers have
rigorously
defined the most primitive stem cells and have provided a description of their
developmental properties. Perhaps the most striking characteristics of this
cell population
come from retrovira) "marking" studies (Leminschka, LR. 1992). These studies
clearly
show that a single stem cell clone is both necessary and sufficient, not only
to sustain
lifelong, multilineage hematopoiesis in one primary recipient but in numerous
secondary
animals. This illustrates the remarkable proliferative potential of the stem
cell and
directly demonstrates stem cell self renewal. A major advance in mouse stem
cell
to biology was the development of strategies which facilitate the substantial
enrichment of
stem cell activity (Bauman, J.G., et al. 1988; Spangrude, G.J., et al. 1988;
Jordan C.T. , et
al 1990). Purification procedures enabled the first direct approaches to
unravel the
mechanisms responsible for the unique biological properties of the stem cell
population.
A key observation was that the phenotypically defined stem/progenitor cell
population is
t5 heterogeneous with respect to in vivo functional properties (Fleming, W.G.,
et al 1993;
Li, C.L. and Johnson G.R. 1992; Spangrude, G.J. and Johnson, G.R. 1990, Jones
R., et
al. 1990; Uchida, N., et al. 1993). In addition to the in vivo repopulating
cells, other
primitive progenitor cells are often contained in a purified population
(Weilbaecher, K.,
1991; Trevisan, M. and Iscove, N.N. 1995; Ogawa, M. 1993). These can be
assayed in a
2o variety of in vitro systems. Whether all of these in vivo and in vitro
activities represent
discrete cell subpopulations or whether there is a continuum of functional
potential is still
an unanswered question. Recent studies have suggested distinct physical
properties for
functionally different activities within the primitive population Mornson,
S.J., and
Weissman, LL. 1994); Mornson, S.J. et al. 1997; Jones, R.M., el al. 1996). A
second set
25 of observations revealed an inverse correlation between a tendency for
active cell cycling
and primitive, uncommitted developmental potential in BM (Spangrude, G.J., and
Johnson, G.R. 1990). In fetal liver a higher proportion of primitive stem
cells is actively
cycling (Fleming, W.H., et al. 1993). Moreover, it has been shown that fetal
stem cells
are more potent than adult stem cells in LTRA (Jordan, C.T., et al. 1995;
Pawliuk, R., et
3o al 1996). These are exciting observations because they suggest that rapid
stem cell
cycling can be compatible with the maintenance of primitive in vivo activity.
Very recent
studies suggest that the adult BM stem cell compartment may in fact be cycling
at a very
slow rate (Bradford, G.B., et al. 1997) . Clearly, stem cell cycle regulation
is a critical


CA 02340465 2001-02-21
WO 00/11168 3 PCT/US99/19052
area for investigation. Stem cell purification has facilitated studies aimed
at ex vivo
maintenance or expansion of the most primitive, transplantable stem cell. Most
culture
systems strongly favor a differentiation process (Van der Sluijs, J.P., et al.
1993;
Traycoff, C.M., et al. 1996; Peters, S.O., et al. 1995; Knobel, K.M., et al.
1994).
However, several recent reports have been encouraging. It has been shown that
colonies
grown in defined cytokines can retain not only myeloid and erythroid but also
lymphoid
potentials (Ball, T.C., et al. 1995). Moreover, the short-term (2-3 weeks)
maintenance of
LTRA has been demonstrated in suspension cultures supported by IL6, IL11,
together
with ckit ligand (KL) or flk2/flt3 ligand (FL) (Yonemura, Y.H., et al. 1997).
A recent
to report has shown that colonies initiated in cytokine-supplemented semisolid
cultures
retain LTRA (Trevisan, M., et al. 1996). The studies described herein have
developed a
stromal cell line supported system which quantitatively maintains LTRA for an
extended
(4-7 weeks) time (Moore, F.A., et al. 1997).
In the human system it is clearly not possible to do the same kind of
extensive
t5 marking and transplantation assays. However, several xenograft model
systems have
been developed to assess the in vivo behavior of human stem cells (Traycoff,
C., et al.
1994; Turner, C., et al. 1996; Cashman, J., et al. 1997) . Some of these
experiments can
be done quantitatively in limiting dilution (Bhatia, M., et al. 1997). A very
recent study
has demonstrated a common provira) integration site in granulocyte macrophages
and T-
2o cells derived from beige/nude/XID mice 7-11 months after engraftment with
genetically
transduced human stem cells (Nolta, J.A., et al. 1996) . This important study
paves the
way for precise in vivo clonal analyses. The largest amount of functional
information
about human stem/progenitor cells has been obtained in vitro using a wide
range of
stromal cell and cytokine supported culture systems. It is not possible herein
to describe
25 and properly accredit all of the important studies, however several
advances deserve
mention. The long-term culture-initiating cell (LTCIC) assay measures the in
vitro
production of colony forming cells (CFC) after periods of at least five weeks
in culture
(Sutherland, H.S., et al. 1989) . The cells producing these CFC derive from a
population
of cells which, at least to some extent, probably overlaps with the most
primitive
3o compartment. The maintenance and expansion of primitive functional
abilities in this
culture system has recently been documented (Petzer, A.L., et al. 1996) . A
variation on
this assay system, the extended LTCIC (ELTCIC) has been suggested to measure
an even
more primitive cell population in BM and CB (Hao, Q.L., et al. 1996) . A very
exciting


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WO 00/11168 4 PCTNS99/19052
prospect for the near future will be the integration of the various in vivo
xenograft assays
with the in vitro LTCIC and ELTCIC systems. Some very recent efforts have
suggested
that the NODSCID xenograft system and the LTCIC assay may measure distinct
stem/progenitor subsets (Larocheile, A..J., et al. 1997). Clearly, much more
work needs
to be done, however, it may be anticipated that the ELTCIC system will provide
the
"bridge" in this continuum. Collectively, and including the various strictly
cytokine-
driven systems, the above studies illustrate the current possibility to
accurately and
quantitatively reveal the majority (if not all) functional entities in the
human
stem/progenitor cell hierarchy. The physical characterization and purification
of human
to stem/progenitor cells has proceeded along lines which are parallel to the
mouse system.
Indeed, because of clinical impetus, it can be argued that they are further
advanced.
Thus, as measured in the range of assays discussed above, the consensus
physical
phenotype of the most primitive portion of the human stem/progenitor hierarchy
is
CD34+Lin-CD38- (Terstappen, L.W.M.M., et al. 1991). The CD34+Lin-CD38+ subset
contains less primitive, more committed cellular entities. Other studies have
shown that,
similar to the mouse, low level expression of Thyl (CD90) is a feature of the
primitive
human stem cell (CD34+Lin-CD90+)(Baum, C.M., et al. 1992; Craig, W., et al.
1993).
Most CD90+ cells in this compartment are CD38-. Therefore, the consensus
phenotype
can be described as CD34+Lin-CD90+ (Craig, W., et al. 1993). Two potential
2o differences with the murine system can now be highlighted. First, a very
recent and
elegant study has shown that the most primitive mouse stem cell may in fact be
CD34-/l0
(Osawa, M., et al. 1996) . Whether this is a genuine difference or whether it
reflects the
ability to perform more accurate long-term engraftment studies in the mouse
remains to
be determined. Second, it has been suggested that in the mouse, CD38
expression is a
positive indicator for primitive stem cell function in a purified population
(Randall, T.D.,
et al. 1996) . As in the mouse, human stem/progenitor cells have been
identified and
purified from various sources. These include: adult BM (Baum, C.M., et al.
1992), CB
(DiGiusto, D.L., et al 1996) , fetal liver (Craig, W., et al. 1993) and
peripheral blood
stem cells after various mobilization protocols (Murray, L., et al. 1994).
Similar to the
3o data obtained in the murine system, comparative studies reveal that, in
general, the basic
and fundamental functional properties of stem/progenitor cells are shared
regardless of
the tissue source. There are however, significant functional and physical
differences.


CA 02340465 2001-02-21
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Numerous insights into hematopoietic molecular control mechanisms have come
from gene-targeting studies in mice. Mutations in specific genes, most
notably, those
encoding transcription or DNA-binding proteins, have profound cell-intrinsic,
global or
lineage-specific effects on hematopoietic development (Shivdasani, R.A., and
Orkin, S.H.
1996; Orkin, S.H. 1996). In the latter cases, it is tempting to speculate that
the phenotypes
result from defects in the commitment process. However, malfunctions in the
commitment decision to "set up" a program of differentiation are difficult to
distinguish
from malfunctions in the differentiation program itself. Two gene products,
AML1
(CBF2) and SCL (tal-1) appear to be necessary global regulators of
hematopoiesis
(Wang, W., et al. 1996; Okuda, T., et al. 1996; Porcher, C., et al. 1996;
Robb, L. , et al.
1996) . Whether these molecules act to specify a hematopoietic stem cell or by
other
means is an open question. Interestingly, both of these molecules play roles
in leukemic
transformation. A very important gain of function study documents the apparent
ability
of HOXB4 to increase primitive cell numbers without significant impairment of
~ 5 differentiation abilities {Sauvageau, G., et al. 1995) . Together with
observations that
HOXA9 is translocated in myeloid leukemia (Nakamura, T., et al. 1996; Borrow,
J., et al.
1996) , these studies suggest an important hematopoietic role for homeobox
proteins.
Without question, the above and other studies have identified important
regulators of
hematopoiesis. However, in almost all cases these regulators were first
identified in other
systems. The opposite approach is to directly search for stem cell regulators
in stem cells,
Graf, L., and Torok-Storb, B. 1995; Yang, Y., et al. 1996). The present
invention solves
these problems.
SUMMARY O~' THE INVENT~nnr
The human hematopoietic stem/progenitor cell population has been extensively
characterized according to physical and antigenic criteria as well as in a
variety of in vitro
and in vivo assay systems. Collectively the human studies have revealed
similarities to
the hierarchical stem/progenitor cell organization defined in the murine
system. In spite
of significant strides in the identification of cytokines which can act on
stem cells, it has
3o not been possible to define a system where undifferentiated expansion of
the most
primitive stem cell population occurs. Similarly, it has not been possible to
direct
differentiation along lineage-specific pathways. These limitations, which also
apply to
the murine system, have hampered the elucidation of regulatory mechanisms
which


CA 02340465 2001-02-21
WO 00/11168 6 PCTNS99/19052
mediate the most fundamental aspect of stem cell biology; that is, the
decision to self
renew or commit to differentiation. As a consequence, very little is known
about the
molecular biology of the most primitive hematopoietic stem cell in any
organism. It was
hypothesized that the regulation of primitive stem cells will be mediated at
least in part by
the products of genes which are uniquely or predominantly expressed in these
cells. One
precedent for an important, differentially-expressed molecule is the flk2/flt3
receptor
tyrosine kinase. There presumably are other important and differentially
expressed gene
products. Therefore, it is an object of the present invention to identify
these molecules
and address their functional roles. Specifically, an aspect of identification
of gene
to expression patterns specific to primitive human stem cells is the molecular
phenotype of
the human stem cell. The present invention describes methods to define the
profile of
genes specifically expressed in undifferentiated human stem/progenitor cell
populations.
A primary focus of the present invention is on primitive cells isolated from
normal
bone marrow (BM) samples. The present invention further comprehends use of
other
t5 sources of stem cells, such as umbilical cord blood (CB).
The methods of the present invention combine diverse technical approaches and
sophisticated bioinformatic analyses.
This invention further provides methods to identify genes whose expression can
be modulated by cytokine or stromal-dependent culture and/or by cell-cycle
status.
2o Another object of the present invention is to provide methods for the
functional
characterization of human stem cell-specific gene products. An aspect of this
invention is
a method to facilitate the functional characterization of specifically
expressed gene
products as candidate regulators of a variety of stem/progenitor cell
processes. In
particular, a provided method uses an in vitro system which approximates many
25 characteristic properties of normal stem cells to analyze positive and
negative regulation
of proliferation, cell-cycle parameters, apoptosis and commitment.
It is a further goal of the present invention to provide a necessary (and
usually
missing) component for stem cell gene-expression screens; that is, the ability
to quickly
assess the function of extensive panels of genes.
3o It is also an object of the present invention to provide a method for the
functional
identification of stem cell regulators. An aspect of this invention is a
facile screening
method for "categorizing" large populations of specifically-expressed
molecules
according to their potential roles in a variety of stem/progenitor cell
processes. Gain of


CA 02340465 2001-02-21
WO 00/1116$ 7 PCT/US99/19052
function as well as loss of function approaches are contemplate by the present
invention.
This method does not rely on any prior knowledge of nucleotide or predicted
protein
sequence.
Accordingly, the present invention provides an isolated nucleic acid
derived from an isolated hematopoietic stem cell, the isolated nucleic acid
comprising the
following characteristics: (1) specifically expressed in the hematopoietic
stem cell; and
{2)
encoding a hematopoietic stem cell - specific protein.
The present invention additionally provides an isolated hematopoietic stem
cell
to specific protein or a portion thereof encoded by the provided nucleic acid.
The present
invention further still provides a nucleic acid probe capable of specifically
hybridizing
with the provided nucleic acid under standard hybridization conditions.
Also, the present invention provides an antibody capable of specifically
binding to
the provided protein without substantially cross-reacting with a non-stem cell
specific
is protein or homologs thereof under conditions permissive to antibody
binding.
Additionally, the present invention provides a cell capable of producing the
provided
antibody.
In addition, the present invention provides a method for identifying the
presence
of a primitive hemopoietic stem cell in a sample comprising nucleic acids
specifically
2o expressed in hematopoietic stem cells. Further still, the present invention
provides a
method for generating a stem cell/progenitor cell from a primitive
hematopoietic cell in a
sample.
The present invention further provides a method for identifying the presence
in a
sample of a compound that modulates hematopoietic stem cell activity.
25 The present invention even further still provides a method for identifying
primitive hematopoietic stem cell-specific nucleic acids.
Also the present invention additionally provides a molecularly defined
primitive
hematopoietic stem cell.
Yet additionally, the present invention provides a method for treating a
condition
3o in a subject comprising administering to the subject a therapeutically
effective amount of
a provided pharmaceutical composition. The present invention provides a method
of
introducing an exogenous nucleic acid into a hematopoietic stem cell.


CA 02340465 2001-02-21
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Finally, the present invention provides a method of ex vivo expansion of
hematopoietic
stem cells. The expanded cells are available to receive exogenous genes,
including by
retroviral or other vectors which require a round of replication.
Alternatively, the cells
are available for transplantation either autologously or heterologously.
FIGURE 1. Schematic "flow of information" strategy.
F1GURE 2. Mammalian stem cell system (black circle = strong hybridization
signal,
gray circle - detectable hybridization signal, and open circle - no detectable
hybridization signal). The Smc-34 cDNA is a completely novel sequence with a
predicted ieucine zipper and several potential membrane spanning domains.
FIGURES 3A-3B. (right panel), the control, non-subtracted RDA cDNA population
(38-
contains b-actin sequences which are missing in the two subtracted RDA
populations,
38- and 38-)38+. A differentially expressed gene (HDD-2, described below) is
enriched
in the 38- RDA population and at least retained in the 38-)38+ RDA population
(Figure
3A, left panel). Two, bi-directional, RDA cDNA populations (38- and the
converse 34
)38) were used to probe (See Figure 3) duplicate arrays of a subtracted 38-
library (Figure
3B).
FIGURE 4A-4E. Figure 4A: 34B4 (SEQ.ID.No.: G9) is closely related to a gene
encoding TINUR The sequence homologies and restricted expression pattern of
34A5 is
shown in Figures 4B and 4C. In Figure 4C and 4D {and also Sa, Sb, and 6b)
there are
twenty-one samples of capfinder-amplified cDNA from various hematopoietic
populations. From left to right these are: four CD34+Lin- populations, three
CD34+Lin-
CD90+ populations, two CD34- populations, four CD34+Lin-CD38+ (obtained from
the
3o same BMs as the CD38- samples in lanes 1-4), two CD34+Lin- samples
(obtained from
the same BMs as the CD90+ samples in lanes 5 and 6), three CD34+Lin-
populations
obtained after 1, 2 or 4 days of culture and finally their three CD34+Lin-
CD38+
counterparts. The 3862 cDNA (SEQ.ID.No.: 70) is closely homologous to the


CA 02340465 2001-02-21
WO 00/11168 9 PCT/US99/19052
LTG9/MLLT3 gene located on 9q22 and involved in t(9;11 ) leukemia (Iida, S.,
et al.
1993) (Figure 4E).
FIGURES SA-5B. GOS3, a fos-related gene (Heximer, S., et al. 1996) (Figure SA)
and
(2) HLA-DR (Figure SB).
FIGURES 6A-6D. Figure 6A. HDD-2 is about 500 bp. It contains a short open
reading
frame of 89 amino acids (SEQ.ID.No.: 71 ). The predicted peptide sequence is
shown.
Figure 6B. The expression profile of HDD-2 demonstrates that it is stem cell
restricted.
o Figure 6C. HDD-2 hybridization to a dot blot with numerous human pA+ mRNA
samples (Clontech). HDD-2 hybridization is only visible in kidney (the other
"spots" are
background). Figure 6D. Genomic Southern blot confirmed that HDD-2 corresponds
to
a single-copy human gene.
FIGURE 7. Immunoprecipitation analysis of protein extracts using rat IgG2b
isotype
control antibody (IgG) or AA4.1 mAb. Protein extracts were prep-ared from D2N
cells;
M2.4 cells; AA4-depleted fetal liver cells (FLAA4-); AA4enriched fetal liver
cells
(FLAA4+); AA4-depleted bone marrow cells (BMAA4-); AA4-enriched bone marrow
cells (BMAA4+). Indicated on the right are positions of molecular weight
markers.
zo
FIGURES 8A-8C. AA4 expression in retrovirus infected cells. Figure 8A. Flow
cytometry analysis of NIH 3T3 and EML Cl cells using PE-conjugated AA4.1
antibody
before and after infection with REBNA/AA4. ~jhure . Imrnunoprecipitation of
biotin-labeled surface proteins using rat IgG2b isotype control antibody
(lanes 1, 3, 5) or
AA4.1 mAb (lanes 2, 4, 6). Cellular extracts were prepared from D2N cells
(lanes 1 and
2), REBNA/AA4 infected NIH 3T3 fibroblasts (lanes 3 and 4), and REBNA/AA4
infected EML Cl cells (lanes 5 and 6). Figure 8C. Immunoprecipitation of
cellular
extracts using AA4.1. REBNA/AA4 NIH 3T3 cells (lanes 2 -- 4) and REBNA/AA4 EML
CI cells (lanes 6 -- 8) were labeled with 35S-methionine and chased with
nonradioactive
media for 10 min. (lanes 3 and 7) or 20 min. (lanes 4 and 8). REBNA/GFP NIH
3T3
(lane 1 ) and REBNA/GFP EML CI cells (lane 5) are shown as controls. Indicated
on the
right are positions of molecular weight markers.


CA 02340465 2001-02-21
WO 00/11168 t0 PCT/US99/19052
FIGURF~9. Nucleotide sequence (SEQ.ID.No.: 72) and the deduced amino acid
sequence of AA4 (SEQ.ID.No.: 73). Amino acid sequences is numbered 1 with
initiator
methionine. Underlined is the putative signal peptide in AA4. The
transmembrane
domain is double underlined. Light grey line indicates C-type lectin
carbohydrate
recognition domain. Dark grey lines show EGF-like repeats, while broken grey
lines
indicate calcium-binding EGF motifs.
FIGURES l0A-IOB. Comparison of the primary structures of AA4 and human CIqR.
_Fiaeure 10A. Alignment of amino acid sequences of AA4 (SEQ.ID.No.: 73) and
CIqR.
to Figure. Comparison of the domain structures of AA4 and CIqR. The proteins
contain N-terminal signal peptides (SP), a C-type lectin recognition domain,
six EGF-like
domains, including three calcium-binding (cb) EGF-like domains, and a
transmembrane
domain (TM).
F1GURE 11. In normal tissues and transformed cell lines, a 7kb RNA species
hybridizes
with the cloned cDNA (Figures I 1 A and 1 I B). In addition to the 7kb
species, poly(A)-
RNA from D2N cells contains a minor band corresponding to a 3.2 kb mRNA
(Figure
I 1 A, lane 8).
2o F1GURES 12A-12B Northern blot analysis of AA4 expression in transformed
cell lines
Figure 12A and normal mouse tissues Figure 12B. Indicated on the left are
positions
of 9.44 - 0.24 kb RNA molecular weight markers. Hybridization with D2N
poly(A)+
RNA is shown after a 2 hr and overnight exposures. Hybridizations with GAPDH
are
shown as controls for equal loading.
FIGURES 13A-13C. RT-PCR of cDNAs prepared from murine fetal liver, (A) bone
marrow-derived hematopoietic cells (B), or differentiating ES cells (C). F~ure
1,~A.
Lanes 1 and 2, AA4- cells; lane 3, AA4+ cells; lane 4, AA4+Sca-1+c-Kit+LinlO
cells;
lane 5, AA4+Sca-1-c-Kit+LinIO cells. Figure 13B. Lane 1, RholOSca-1+Thy-
1 IOLincells; lane 2, RhohiSca-1+Thy-1 lOLin- cells; lane 3, Sca-I+Thy-l-Lin-
cells; lane
4, Lin+ cells. Figure 13C_. ESO, undifferentiated ES cells; BLI and 2, blast
cell colonies;
ENT1 and 2, differentiated endothelial cells; HMT1, 2 and 3, differentiated
hematopoietic
cells. The cDNAs probes used for hybridization are indicated on the right.


CA 02340465 2001-02-21
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FIGURE 14. AFT024 maintains in vivo repopulating stem cells. The ability of 3
different stromal cell lines to support highly purified fetal liver stem cells
was studied.
Freshly purified day 14 fetal liver cells were transplanted directly (103
AA4.1+, lin'~°,
s Sca-1+, c-kit+ cells plus lOG Ly5.1 competitor BM per mouse; n=6) into Ly5.1
congenic
mice (Control). Additional stem cells from the same purification were also
used to
initiate Dexter-LTC over inradiated AFT024, 2012, and 2018 stromal cell
monolayers
(104 cells/10 cm dish}. After 4 weeks of culture, 10% aliquots of each culture
(103 stem
cell equivalents) were transplanted into Ly5.1 recipients (n=8) together with
10~'
to competitor Ly5.1 BM cells. Peripheral blood cells from mice were analyzed
for the
presence of Ly5.2~ cells at S, 12, 24, and 56 weeks after transplant. Error
bars represent
SEM.
FIGURE 15. Long-term culture on AFT024 maintains greater levels of
repopulating
15 stem cell activity than short-term cytokine- or short-term AFT024-supported
cultures.
The levels of stem cell activity maintained in short-term cytokine-supported
and short-
term AFT024-supported cultures were compared to those maintained in long-term
AFT024 coculture. Purified fetal liver cells were cultured for 5 days in
suspension with
cytokines or on an AFT024 monolayer (3000/well-12-well tray). Additional cells
from
20 the same purification were seeded onto AFT024 monolayers (3000/6 cm dish)
and
maintained in Dexter-LTC for 5 weeks. At completion of both culture periods
the cells
were harvested, mixed with Ly5.1 BM and used to transplant mice. Each mouse
received
20% of each culture (600 stem cell equivalents) and 4X105 competitor BM cells
(4
mice/culture). Peripheral blood cells from mice were analyzed for the presence
of Ly5.2+
2s cells at 15 weeks after transplant. FL 1.0+0.57; FL/SL 0.75+0.25; FL/IL-6
1.8+0.14;
SL/IL-6 3.2+p.46; FL/IL-6/SL 1.7~0.2I; AFT024 5 days 2.8+0.11; AFT024 5 weeks
32.2~7.4 * (p<p.004) Students t-test. See Methods for culture conditions and
cytokine
concentrations. FL, flk2/flt3-ligand; SL, steel factor. Error bars represent
SEM.
3o FIGURE 16. Time course of cobblestone area formation on AFT024. The
formation of
stromal dependent CA derived from purified fetal liver stem cells was studied
in AFT024
cocultures. Characteristic clusters of at least 50 cells were scored as CA
over 28 days of
culture. Results are expressed as the mean number of CA/1000 input stem cells
from 3


CA 02340465 2001-02-21
WO 00/11168 12 PCTNS99/19052
separate fetal liver purifications (300-600 cells/well in 12-well trays).
Error bars
represent the SEM. The frequency of CA after 28 days is approximately 1 for
every 20
input stem cells.
FIGURE 17. High-proliferative potential multilineage clonogenic progenitors
are
selectively expanded on AFT024. The clonogenic progenitor content of stem
cells
maintained in AFT024 supported Dexter-LTC was deternnined. Enriched fetal
liver stem
cells were seeded onto AFT024 monolayers, at various time points, an
individual well
was harvested and the cells placed into semi-solid clonogenic progenitor assay
(CFU-C).
to Colonies were scored at 8-14 days. Colonies were designated as HPP upon
reaching a
size __>1 mm after 8 days. CFU numbers at days 0, 4, and 28 are averaged from
3-5
individual stem cell purifications. Error bars represent the SEM for these
experiments.
Other time points are individual determinations. CFU are normalized to 1000
input stem
cells in the stromal cocultures for comparison to day 0 progenitors. *CFU-Mix
(p=.O1 )
t5 and *CFU-HPP-Mix (p=.001) are significantly expanded at day 28 compared to
day 0,
(Student's T-test).
FIGURES 18A-18B. Cobblestone area-initiating cells are expanded on AFT024.
Figure 18A. A quantitative estimate of the number of 28 day cultured stem cell
20 equivalents required to form a CA after replating on secondary AFT024
monolayers was
determined. Four different AFT024 cocultures from separate fetal liver
purifications
were studied (A, B, C, and D). In limiting-dilution assay, the frequency of
stem cell
equivalents required to form a CA in another 7 days was 1 in 4 (3.56+0.64,
r2=0.96).
Figure 18 B. CA maintenance in one of the 4 above cultures was followed for an
25 additional 4 weeks. The frequency of stem cell equivalents maintaining CA
was
determined. At 37% negative wells the frequencies were: 2 weeks 1 in 3, 3
weeks 1 in
10, 4 weeks 1 in 19, and at 5 weeks 1 in 29.
FIGURES 19A-19B. Dlk expression analysis in stromal cell lines. Figure 19A.
(Top) A
30 1.6 kb dlk transcript is visualized in the parental AFT024 and 2012 cell
lines and their
subclones, but not in 2018, CFC034 and BFC012. (Bottora) The same filter
hybridized with
a b-actin probe. Figure 19B. RT-PCR analysis of 14 fetal liver-derived stromal
cell lines
and other cell lines.


CA 02340465 2001-02-21
WO 00/11168 13 PCTNS99/19052
FIGURE 20. Cobblestone area formation by hematopoietic stem cells in the
presence of
soluble dlk protein. Data are from 4 experiments; 2 each with adult BM cells
(Sca-1+, c-
kit+, line°~-) and day 14 fetal liver cells (AA4.1+, Sca-1+, c-kit+,
line°~-). Results are expressed
as the ratio/fold-increase in CSA number for fourteen data points each (bars
represent, none
vs control; dlk vs none; dlk vs control) for the 4 different experiments.
Error bars represent
SEM. *P= 0.01 comparing dlk vs none to none vs control, **P= 0.001 comparing
dlk vs
control to none vs control (Student's t-test).
to FIGURE 21. Membrane bound dlk expression in transfected BFC012 cells. Full
length dlk
cDNA was transfected into BFC012 cells. (Left) A flow diagram of dlk
expression in
transfected BFC012 populations (BFC-dlk) and cells transfected with the
selection plasmid
alone (BFC-Zeo). (Right) Expression of dlk in a cloned line (BFC-dlk-5)
derived from the
expressing population and a control clone (BFC-Zeo-1 ).
FIGUR .~ 22A, CSA formation by hematopoietic stem cells in the presence of
membrane-
bound dlk. Figure 22A. Bars labeled BFC are from 5 groups (nontransfected
BFC012 cells,
two control pZeo transfected BFC012 populations, and two clones derived from
the pZeo
transfected populations). Bars labeled BFC-dlk are from three groups shown to
express
2o transfected dlk; one dlk-transfected BFC012 population (BFC-dlk) and two
individual
transfected clones (BFC-dlk-1 and BFC-dlk-5). Error bars represent SEM. ** P<
0.001
days 3, 4, and 5; *P< 0.01 days 6 and 7 (Student's t-test). Figure 22B. A
clone derived
from the dlk transfected populations of BFC012 cells (BFC-dlk-5) and a clone
derived from
pZeo transfected populations (BFC-Zeo-1 ) were used for CSA assay with
purified fetal liver
stem cells. CSA/1000 input stem cells are expressed as the mean of three
individual
experiments, error bars represent the SEM. **P< 0.001 at days 4, G, and 8
(Student's t-test).
IfIGURES 23A-2'~R f-lpp multilineage clonogenic progenitors and irr vivo
repopulating
stem cells are maintained in short-term dlk-expressing cocultures. Figure 23A.
Fetal liver
stem cells were purified as described and assayed for their progenitor content
immediately
after purification and after culture on BFC-dlk-5, and BFC-Zeo-1. At day 4 the
cultures
were used for clonogenic progenitor (3 experiments) and transplantation assay
(2
experiments). Bars represent data from 3 experiments with day 0 cells (Fresh)
and day 4


CA 02340465 2001-02-21
WO 00/11168 14 PCT/US99/19052
cocultured cells (BFC-dlk-5 and BFC-Zeo-1 ), error bars represent SEM. *P=
0.01 for total
CFU-C from fresh stem cells compared to total CFU-C from BFC-dlk-5 cocultures
at day 4;
** P= 0.001 for total CFU-C from BFC-dlk-5 compared to total CFU-C from BFC-
Zeo-1
(Student's t-test). Figure 23 B. Analysis of in vivo repopulating ability of
purified fetal
liver stem cells cocultured for 4 days on BFC-dlk-5, BFC-Zeo-1, and AFT024
monolayers.
Results are from nine individual mice in two experiments (4-5 mice in each
experiment) at
weeks after transplantation. P= 0.05 for BFC-dlk-5 vs BFC-Zeo-I (Student's t-
test).
FIGURE 24. Genes and predicted proteins isolated from primitive stem cells by
the
o techniques of the present invention. (Sequence Identification Numbers are
indicated on the
figure).
DETAILED DE~RmTmN
The present invention provides an isolated nucleic acid derived from an
isolated
hematopoietic stem cell, the isolated nucleic acid comprising the following
characteristics: (1) specifically expressed in the hematopoietic stem cell;
and (2)
encoding a hematopoietic stem cell - specific.protein.
An embodiment of this invention further comprises the following
characteristic:
2o capable of hybridizing under standard conditions with a sequence selected
from the group
consisting of SEQ.ID.No.: 1, SEQ.ID.No.: 2, SEQ.ID.No.: 3, SEQ.ID.No.: 4,
SEQ.ID.No.: 5, SEQ.ID.No.: G, SEQ.ID.No.: 7, SEQ.ID.No.: 8, SEQ.ID.No.: 9,
SEQ.ID.No.: 10, SEQ.ID.No.: 11, SEQ.ID.No.: 12, SEQ.ID.No.: 13, SEQ.ID.No.:
14,
SEQ.ID.No.: 15, SEQ.ID.No.: IG, SEQ.ID.No.: 17, SEQ.ID.No.: 18, SEQ.ID.No.:
19,
SEQ.ID.No.: 20, SEQ.ID.No.: 21, SEQ.ID.No.: 22, SEQ.ID.No.: 23, SEQ.ID.No.:
24,
SEQ.ID.No.: 25, SEQ.ID.No.: 2G, SEQ.ID.No.: 27, SEQ.ID.No.: 28, SEQ.ID.No.:
29,
SEQ.ID.No.: 30, SEQ.ID.No.: 31, SEQ.ID.No.: 32, SEQ.ID.No.: 33, SEQ.ID.No.:
34,
SEQ.ID.No.: 35, SEQ.ID.No.: 36, SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.:
39,
SEQ.ID.No.: 40, SEQ.ID.No.: 41, SEQ.ID.No.: 42, and SEQ.ID.No.: 43,
SEQ.ID.No.:
45, SEQ.ID.No.: 47, SEQ.ID.No.: 49, SEQ.ID.No.: 51, SEQ.ID.No.: 53,
SEQ.ID.No.: 55,
SEQ.ID.No.: 57, SEQ.ID.No.: 59, SEQ.ID.No.: GI, SEQ.ID.No.: G3, SEQ.ID.No.:
65,
SEQ.ID.No.: G7, SEQ.ID.No.: 72 or a portion thereof. A portion thereof, in a
preferred
embodiment of this invention is the 5' end region or the 3' end region of the
nucleic acid.


CA 02340465 2001-02-21
WO 00/11168 15 PCT/US99/19052
In another preferred embodiment, a portion thereof is at least a 8-18
nucleotide portion of
the coding region. In yet another preferred embodiment, a portion therof is at
least a 8-18
nucleotide portion of a non-coding regulatory region or a binding region such
as a stem-
cell specific promoter or enhancer region. According to still another
embodiment of the
provided isolated nucleic acid further comprises the characteristic of
encoding a protein
capable of modulating hematopoietic stem cell activity. According to this
invention
modulating hematopoietic stem cell activity includes up-regulating, down-
regulating or
otherwise changing the activity of the hematopoietic stem cell. Such activity
is
contemplated as inducing differentiation or inhibiting differentiation of the
cell.
to However, directing differentiation toward one or another daughter cell type
is also within
the scope of a preferred embodiment of this invention. Other preferred
embodiments
include but are not limited to modulation of transcription, translation, gene
splicing,
transport, proteolytic processing, replication, expression of cell surface
markers and
transplantation. According to still another embodiment of the present
invention, the
activity is selected from the group consisting of hematopoietic stem cell
differentiation
and hematopoietic stem cell replication. According to yet another embodiment
of this
invention, the protein is selected from the group consisting of a growth
factor, a
transcription factor, a splicing factor, a capping factor, a transport
protein, a translation
factor, and a replication factor. In one preferred embodiment of this
invention, the
provided nucleic acid comprises the nucleotide sequence of SEQ.ID.No.: 72, an
analog
thereof, or a portion thereof. According to another preferred embodiment of
this
invention, the hematopoietic stem cell is a primitive hematopoietic stem cell.
In one
embodiment of this invention, the primitive hematopoietic stem cell is
selected from the
group consisting of an umbilical cord cell , a bone marrow cell and a fetal
liver cell. In a
preferred embodiment of this invention, the primitive hematopoietic stem cell
is selected
from the group consisting of a AFT024 cell, a 2012 cell and a 2018 cell.
The present invention further provides a composition comprising the provided
nucleic acid, wherein the nucleic acid comprises one selected from the group
consisting
of SEQ.ID.No.: 1, SEQ.ID.No.: 2, SEQ.ID.No.: 3, SEQ.ID.No.: 4, SEQ.ID.No.: S,
3o SEQ.ID.No.: 6, SEQ.ID.No.: 7, SEQ.ID.No.: 8, SEQ.ID.No.: 9, SEQ.ID.No.: 10,
SEQ.ID.No.: 11, SEQ.ID.No.: 12, SEQ.ID.No.: 13, SEQ.ID.No.: 14, SEQ.ID.No.:
15,
SEQ.ID.No.: 1 G, SEQ.ID.No.: 17, SEQ.ID.No.: 18, SEQ.ID.No.: 19, SEQ.ID.No.:
20,
SEQ.ID.No.: 21, SEQ.ID.No.: 22, SEQ.ID.No.: 23, SEQ.ID.No.: 24, SEQ.ID.No.:
25,


CA 02340465 2001-02-21
WO 00/11168 16 PCT/US99/19052
SEQ.ID.No.: 26, SEQ.ID.No.: 27, SEQ.ID.No.: 28, SEQ.ID.No.: 29, SEQ.ID.No.:
30,
SEQ.ID.No.: 31, SEQ.ID.No.: 32, SEQ.ID.No.: 33, SEQ.ID.No.: 34, SEQ.ID.No.:
35,
SEQ.ID.No.: 36, SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.: 39, SEQ.ID.No.:
40,
SEQ.ID.No.: 41, SEQ.ID.No.: 42, SEQ.ID.No.: 43, SEQ.ID.No.: 45, SEQ.ID.No.:
47,
SEQ.ID.No.: 49, SEQ.ID.No.: 51, SEQ.ID.No.: 53, SEQ.ID.No.: 55, SEQ.ID.No.:
57,
SEQ.ID.No.: 59, SEQ.ID.No.: 61, SEQ.ID.No.: 63, SEQ.ID.No.: 65, SEQ.ID.No.:
67,
SEQ.ID.No.: 72 or a portion thereof. According to one embodiment of this
invention, the
the nucleic acid is selected from the group consisting of DNA, RNA and cDNA.
Another
embodiment of this invention is a vector comprising the provided nucleic acid.
to According to yet another embodiment, the vector comprises viral or plasmid
DNA. A
further embodiment of this invention is an expression vector comprising the
provided
nucleic acid and a regulatory element. A still further embodiment of this
invention is a
host vector system which comprises the expression vector in a suitable host.
In a
preferred embodiment of this invention, the suitable host is selected from the
group
t S consisting of a bacterial cell, a eukaryotic cell, a mammalian cell and an
insect cell.
The present invention additionally provides an isolated hematopoietic stem
cell
specific protein or a portion thereof encoded by the provided nucleic acid.
According to
one embodiment of this invention is the provided protein further comprising
the
characteristic of being capable of modulating hematopoietic stem cell
activity. According
20 to this invention modulating hematopoietic stem cell activity includes up-
regulating,
down-regulating or otherwise changing the activity of the hematopoietic stem
cell. Such
activity is contemplated as inducing differentiation or inhibiting
differentiation of the cell.
However, directing differentiation toward one or another daughter cell type is
also within
the scope of a preferred embodiment of this invention. Other preferred
embodiments
25 include but are not limited to modulation of transcription, translation,
gene splicing,
transport, proteolytic processing, replication, expression of cell surface
markers and
transplantation. According to still another embodiment of the present
invention, the
activity is selected from the group consisting of hematopoietic stem cell
differentiation
and hematopoietic stem cell replication. According to yet another embodiment
of this
3o invention, the protein is selected from the group consisting of a growth
factor, a
transcription factor, a splicing factor, a capping factor, a transport
protein, a translation
factor, and a replication factor. According to one embodiment the activity is
selected
from the group consisting of hematopoietic stem cell differentiation and
hematopoietic


CA 02340465 2001-02-21
WO 00/11168 t ~ PCTNS99/19052
stem cell replication. According to another embodiment, the protein is
selected from the
group consisting of a growth factor, a transcription factor, a splicing
factor, a capping
factor, a transport protein, a translation factor, and a replication factor.
According to still
another embodiment, the protein has substantially the same amino acid sequence
as one
selected from the group consisting of SEQ.ID.No.: 42, SEQ.ID.No.: 44,
SEQ.ID.No.: 46,
SEQ.ID.No.: 48, SEQ.ID.No.: 50, SEQ.ID.No.: 52, SEQ.ID.No.: 54, SEQ.ID.No.:
56,
SEQ.ID.No.: 58, SEQ.ID.No.: 60, SEQ.ID.No.: 62, SEQ.ID.No.: 64, SEQ.ID.No.:
66,
SEQ.ID.No.: 68, SEQ.ID.No.: 70, SEQ.ID.No.: 71, and SEQ.ID.No.: 73.
The present invention further still provides a nucleic acid probe capable of
to specifically hybridizing with the provided nucleic acid under standard
hybridization
conditions.
Also, the present invention provides an antibody capable of specifically
binding to
the provided protein without substantially cross-reacting with a non-stem cell
specific
protein or homologs thereof under conditions permissive to antibody binding.
Additionally, the present invention provides a cell capable of producing the
provided
antibody.
In addition, the present invention provides a method for identifying the
presence
of a primitive hemopoietic stem cell in a sample comprising nucleic acids
specifically
expressed in hematopoietic stem cells comprising (a) contacting the sample
with the
2o provided antibody under conditions permissive to the formation of an
antibody complex;
(b) detecting the presence of the complex formed in step (a), the presence of
a complex
formed indicating the presence of a primitive hemopoietic stem cell in the
sample.
According to one embodiment of this invention, the antibody is labeled with a
detectable
marker. In a preferred embodiment, the detectabie marker is selected from the
group
consisting of a radioactive isotope, enzyme, magnetic bead, dye, flourescent
marker and
biotin.
Further still, the present invention provides a method for generating a stem
cell/progenitor cell from a primitive hematopoietic cell in a sample
comprising contacting
the sample with the provided protein . Another embodiment of this invention,
provides a
3o method for generating a stem cell/progenitor cell from a primitive
hematopoietic cell in a
sample comprising contacting the sample with the provided nucleic acid.
According to an
preferred embodiment, the nucleic acid is in an expression vector. According
to another
preferred embodiment the nucleic acid is introduced into the cell under
conditions


CA 02340465 2001-02-21
WO 00/11168 i g PCT/US99/19052
permissive to the expression of the nucleic acid.
The present invention further provides a method for identifying the presence
in a
sample of a compound that modulates hematopoietic stem cell activity
comprising: (a)
contacting the hematopoietic stem cell with the sample; (b) determining the
hematopoietic stem cell activity; and (c) comparing the hematopoietic stem
cell activity
determined in step (b) with the activity determined in the absence of the
compound an
increase or decrease in hematopoietic stem cell activity indicating the
presence in the
sample of a compound that modulates hematopoietic stem cell activity.
According to one
embodiment, the activity is selected from the group consisting of gene
expression,
o replication, differentiation, transplantation, and self regeneration. The
present invention
also still further provides a compound identified by the method of this
invention,
previously unknown.
The present invention even further still provides a method for identifying
primitive hematopoietic stem cell-specific nucleic acids , comprising: (a)
creating a
primitive hematopoietic stem cell cDNA library and a non-primitive stem cell
immune
cell cDNA library; and (b) subtracting the two libraries, thereby identifying
primitive
stem cell specific nucleic acids. According to one embodiment is (i)
contacting the
nucleic acids of the stem cell and non-stem cell libraries with each other
under conditions
permissive to hybridization, thereby forming hybrid complexes; (ii) separating
the hybrid
zo complexes formed in step (b) from the nucleic acids which did not form
complexes; and
(iii) isolating the nucleic acids which did not form complexes, thereby
identifying
hematopoietic stem cell specific nucleic acids. In still another embodiment,
step (ii)
further comprising amplification of the nucleic acids. Yet another embodiment
is step
(iii) further comprising ampliciation of the nucleic acids which did not form
complexes.
Even still another embodiment is further comprising displaying the amplified
DNA on a
chromatography gel. A further embodiment is step (b) comprising differential
display of
the two libraries, thereby identifying primitive stem cell specific nucleic
acids. Also yet
another embodiment is step (b) comprising representation difference analysis
of the two
libraries, thereby identifying primitive stem cell specific nucleic acids. Yet
even another
3o embodiment is further comprising cloning the stem cell specific nucleic
acid. According
to a preferred embodiment, the stem cell is selected from the group consisting
of AF024,
2012, and 2018. The present invention further provides a nucleic acid
identifted by the
provided method.


CA 02340465 2001-02-21
WO 00/11168 19 PCT/US99/19052
The present invention additionally provides a composition comprising the
provided compound and a carrier. An embodiment of this invention is a
pharmaceutical
composition comprising the provided compound and a pharmaceutically acceptable
carrier.
s Also the present invention additionally provides a primitive hematopoietic
stem
cell specifically expressing one selected from the group consisting of:
SEQ.ID.No.: I,
SEQ.ID.No.: 2, SEQ.ID.No.: 3, SEQ.ID.No.: 4, SEQ.ID.No.: 5, SEQ.ID.No.: 6,
SEQ.ID.No.: 7, SEQ.ID.No.: 8, SEQ.ID.No.: 9, SEQ.ID.No.: 10, SEQ.ID.No.: Il,
SEQ.ID.No.: 12, SEQ.ID.No.: 13, SEQ.ID.No.: 14, SEQ.ID.No.: 15, SEQ.ID.No.:
16,
to SEQ.ID.No.: 17, SEQ.ID.No.: 18, SEQ.ID.No.: 19, SEQ.ID.No.: 20, SEQ.ID.No.:
21,
SEQ.ID.No.: 22, SEQ.ID.No.: 23, SEQ.ID.No.: 24, SEQ.ID.No.: 25, SEQ.ID.No.:
26,
SEQ.ID.No.: 27, SEQ.ID.No.: 28, SEQ.ID.No.: 29, SEQ.ID.No.: 30, SEQ.ID.No.:
31,
SEQ.ID.No.: 32, SEQ.ID.No.: 33, SEQ.ID.No.: 34, SEQ.ID.No.: 35, SEQ.ID.No.:
36,
SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.: 39, SEQ.ID.No.: 40, SEQ.ID.No.:
41,
is SEQ.ID.No.: 42, and SEQ.ID.No.: 43, SEQ.ID.No.: 45, SEQ.ID.No.: 47,
SEQ.ID.No.:
49, SEQ.ID.No.: 51, SEQ.ID.No.: 53, SEQ.ID.No.: 55, SEQ.ID.No.: 57,
SEQ.ID.No.: 59,
SEQ.ID.No.: 61, SEQ.ID.No.: 63, SEQ.ID.No.: 65, SEQ.ID.No.: 67, SEQ.ID.No.: 72
or a
portion thereof. An embodiment of this invention is a primitive hematopoietic
stem cell
specifically expressing a nucleic acid identified by the provided method.
20 Yet additionally, the present invention provides a method for treating a
condition
in a subject comprising administering to the subject a therapeutically
effective amount of
the provided pharmaceutical composition. According to an embodiment of this
invention,
the condition is an immune system condition. In a further embodiment of this
invention,
the condition is leukemia.
25 The present invention provides a method of introducing an exogenous nucleic
acid
into a hematopoietic stem cell comprising contacting the stem cell with the
provided
composition.
Finally, the present invention provides a method of ex vivo expansion of
hematopoietic stem cells comprising contacting the cell with the provided
composition.
3o According to an embodiment of this invention, the ex vivo expanded
hematopoietic stem
cells are available for therapeutic use. The expanded cells are available to
receive
exogenous genes, including by retroviral or other vectors which require a
round of


CA 02340465 2001-02-21
WO 00/11168 20 PCT/US99/19052
replication. Alternatively, the cells are available for transplantation either
autologously or
heterologously.
As used herein, the term, a sequence is conserved if there is substantial
homology
of sequence between multiple gene species.
As used herein, the terms, "hybridization" and "binding" in the context of
probes,
primers and denatured DNA are used interchangeably. Probes which are
hybridized or
bound to denatured DNA are aggregated to complementary sequences in the
polynucleotide. Whether or not a particular probe remains aggregated with the
polynucleotide depends on the degree of complementarity, the length of the
probe, and
to the stringency of the binding conditions. The higher the stringency, the
higher must the
degree of complementarity, and/or the longer the probe.
As used herein, the term, probe, refers to an oligonucleotide designed to be
sufficiently complementary to a sequence in a denatured nucleic acid to be
probed, in
relation to its length, to be bound under selected stringency conditions.
Primers may vary
in length. Preferably such primers should be sufficiently long to hybridize to
the
modified RNAs in a specific and stable manner. A semi-random primer as the
term is
used herein, encompasses a class of primers wherein either a discrete portion
of the
primer is random, while another discrete portion is conserved as well as
primers which
have nucleotide preferences at particular positions within a sequence. For
example, the
2o discrete portion-type primer may have a predetermined adaptor sequence at
its 5' end and
a random sequence at its 3' end. Alternatively, several preferred primers have
nucleotide
preferences at specific positions within the primers while other positions are
random. A
degenerate primer as the term is used herein, encompasses a cocktail or
mixture of
primers wherein one or more of the possible triplet nucleotide sequences
encoding an
amino acid is incorporated into the primer sequence. For example, Serine may
be
encoded by six separate triple sequences (AGU, AGC, UCU, UCC, UCA, and UCG).
Thus, a "degenerate" primer may reflect the degeneracy of the nucleotide
triplet code.
Alternatively, a randomized primer, as the term is used herein, encompasses a
primer
wherein, the nucleotide at one or more positions may be randomized in order to
yield a
3o triplet sequence encoding an alternative or a random amino acid at the
position.
An end region as the term is used herein, consists of the end nucleotide and a
portion of the region including as much as that half of the entire sequence.
For example,
the "3' end region" or "3' region" of a primer may include the 3' half of the
primer.


CA 02340465 2001-02-21
WO 00/11168 21 PCT/US99/19052
A preferred method of hybridization is blot hybridization. See Sambrook et al.
1989 Molecular Cloning: A Laboratory Manual 2nd Ed. for additional details
regarding
blot hybridization. Using this method, separated amplification products are
transferred
onto a solid matrix, such as a filter. The probe, which is detectable, either
directly or
indirectly, is hybridized to the solid matrix under appropriate conditions.
The matrix is
washed to remove excess probe. Thereafter the probe which specifically
hybridized to
the solid matrix can be detected.
The probe can be DNA or RNA and can be made detectable by any of the many
labeling techniques readily available and known to the skilled artisan. Such
methods
to include, but are not limited to, radio-labeling, digoxygenin-labeling, and
biotin-labeling.
A well-known method of labeling DNA 15 3zP using DNA polymerise, Klenow enzyme
or polynucleotide kinase. In addition, there are known non-radioactive
techniques for
signal amplification including methods for attaching chemical moieties to
pyrimidine and
purine rings (Dale, R.N.K. et al, 1973 Proc. Natl. Acid. Sci. USA 70:2238-42),
methods
which allow detection by chemiluminescence (Barton, S.K. et al, 1992 J. Am.
Chem. Soc.
114:8736-40) and methods utilizing biotinylated nucleic acid probes (Johnson,
T.K. et al,
1983 Anal. Biochen:. 133:125-131; Erickson, P.F. et al, 1982 J. Immunol.
Methods
51:241-49; Matthaei, F.S. et al, 1986 Anal. Biochem. 157-123-28) and methods
which
allow detection by fluorescence using commercially available products. Non-
radioactive
labeling kits are also commercially available.
A basic description of nucleic acid amplification is described in Mullis, U.S.
Patent No. 4,683,202, which is incorporated herein by reference. The
amplification
reaction uses a template nucleic acid contained in a sample, two primer
sequences and
inducing agents. The extension product of one primer when hybridized to the
second
2s primer becomes a template for the production of a complementary extension
product and
vice versa, and the process is repeated as often as is necessary to produce a
detectable
amount of the sequence.
The inducing agent may be any compound or system which will function to
accomplish the synthesis of primer extension products, including enzymes.
Suitable
3o enzymes for this purpose include, for example, E.coli DNA polymerise I,
thelmostable
Taq DNA polymerise, Klenow fragment of E.coli DNA polymerise I, T4 DNA
polymerise, other available DNA polymerises, reverse transcriptase and other
enzymes
which will facilitate combination of the nucleotides in the proper manner to
form


CA 02340465 2001-02-21
WO 00/11168 22 PCT/US99/19052
amplification products. The oligonucleotide primers can be synthesized by
automated
instruments sold by a variety of manufacturers or can be commercially prepared
based
upon the nucleic acid sequence of this invention.
The degree of hybridization depends on the degree of complementarity, the
length
of the nucleic acid molecules being hybridized, and the stringency of the
conditions in a
reaction mixture. Stringency conditions are affected by a variety of factors
including, but
not limited to temperature, salt concentration, concentration of the nucleic
acids, length of
the nucleic acids, sequence of the nucleic acids and viscosity of the reaction
mixture.
More stringent conditions require greater complementarity between the nucleic
acids in
order to achieve effective hybridization.
"Hybridization" and "binding" in the context of probes, primers and denatured
DNA are used interchangeably. Probes which are hybridized or bound to
denatured DNA
are aggregated to complementary sequences in the polynucleotide. Whether or
not a
particular probe remains aggregated with the polynucleotide depends on the
degree of
is complementarity, the length of the probe, and the stringency of the binding
conditions.
The higher the stringency, the higher must the degree of complementarity,
and/or the
longer the probe.
"Probe" refers to an oligonucleotide designed to be sufficiently complementary
to
a sequence in a denatured nucleic acid to be probed, in relation to its
length, to be bound
2o under selected stringency conditions.
Primers may vary in length. Preferably such primers should be sufficiently
long
to hybridize to the modified RNAs in a specific and stable manner.
A semi-random primer as the term is used herein, encompasses a class of
primers
wherein either a discrete portion of the primer is random, while another
discrete portion is
25 conserved as well as primers which have nucleotide preferences at
particular positions
within a sequence. For example, the discrete portion-type primer may have a
predetermined adaptor sequence at its 5' end and a random sequence at its 3'
end.
Alternatively, several preferred primers have nucleotide preferences at
specific positions
within the primers while other positions are random.
3o A degenerate primer as the term is used herein, encompasses a cocktail or
mixture
of primers wherein one or more of the possible triplet nucleotide sequences
encoding an
amino acid is incorporated into the primer sequence. For example, Serine may
be
encoded by six separate triple sequences (AGU, AGC, UCU, UCC, UCA, and UCG).


CA 02340465 2001-02-21
WO 00/11168 23 PCT/US99/19052
Thus, a "degenerate" primer may reflect the degeneracy of the nucleotide
triplet code.
Alternatively, a randomized primer, as the term is used herein, encompasses a
primer
wherein, the nucleotide at one or more positions may be randomized in order to
yield a
triplet sequence encoding an alternative or a random amino acid at the
position.
Solid matrices are available to the skilled artisan. Solid phases useful to
serve as a
matrix for the present invention include but are not limited to polystyrene,
polyethylene,
polypropylene, polycarbonate, or any solid plastic material in the shape of
test tubes,
beads, microparticles, dip-sticks, plates or the like. Additionally matrices
include, but are
not limited to membranes, 96-well microtiter plates, test tubes and Eppendorf
tubes.
o Solid phases also include glass beads, glass test tubes and any other
appropriate shape
made of glass. A functionalized solid phase such as plastic or glass which has
been
modified so that the surface carries carboxyl, amino, hydrazide, or aldehyde
groups can
also be used. In general such matrices comprise any surface wherein a iigand-
binding
agent can be attached or a surface which itself provides a ligand attachment
site.
As used herein, "pharmaceutically acceptable" refers to molecular entities and
compositions that are physiologically tolerable and do not typically produce
an allergic
or similar untoward reaction, such as gastric upset, dizziness and the like,
when
administered to a human. A pharmaceutically acceptable carrier encompasses any
of
the standard pharmaceutically accepted carriers, such as phosphate buffered
saline
2o solution, water emulsions such as an oil/water emulsion or a triglyceride
emulsion,
various types of wetting agents, tablets, coated tablets and capsules.
Typically such
carriers contain excipients such as starch, milk, sugar, certain types of
clay, gelatin,
stearic acid, talc, vegetable fats or oils, gums, glycols, or other known
excipients. Such
carriers may also include flavor and color additives or other ingredients. The
invention
2s also provides for pharmaceutical compositions together with suitable
diluents,
preservatives, solubilizers, emulsifiers and adjuvants. Other embodiments of
the
compositions of the invention incorporate particulate forms, protective
coatings,
protease inhibitors or permeation enhancers for various routes of
administration,
including but not limited to intravenous, intramuscular, parenteral,
pulmonary, nasal
3o and oral.
As used herein, an "effective amount" is the amount required to achieve a
clinically significant effect. For example a significant reduction of
infection, or


CA 02340465 2001-02-21
WO 00/11168 24 PCT/US99/19052
reduction of cell growth or reduction of tumor size is a reduction of
preferably of at
least 30 percent, more preferably of at least 50 percent, most preferably of
at least 90
percent. Accordingly, the effective amount will vary with the subject being
treated, as
well as the condition to be treated. For the purposes of this invention, the
methods of
s administration are to include, but are not limited to administration
cutaneously,
subcutaneously, intravenously, parenterally, orally, topically, or by aerosol.
The present invention further contemplates therapeutic compositions useful in
practicing the therapeutic methods of this invention. A subject therapeutic
composition
includes, in admixture, a pharmaceutically acceptable excipient (carrier) and
one or
1 o more of a polypeptide analog or fragment of the provided peptide or
peptide
composition, a peptidomimetic composition thereof as described herein as an
active
ingredient. A cocktail of the provided pharmaceutical composition in various
combinations is also contemplated.
The preparation of therapeutic compositions which contain polypeptides,
analogs
is or active fragments as active ingredients is well understood in the art.
Typically, such
compositions are prepared as injectables, either as liquid solutions or
suspensions,
however, solid forms suitable for solution in, or suspension in, liquid prior
to injection
can also be prepared. The preparation can also be emulsified. The active
therapeutic
ingredient is often mixed with excipients which are pharmaceutically
acceptable and
2o compatible with the active ingredient. Suitable excipients are, for
example, water,
saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In
addition, if
desired, the composition can contain minor amounts of auxiliary substances
such as
wetting or emulsifying agents, pH buffering agents which enhance the
effectiveness of
the active ingredient.
25 A polypeptide, analog or active fragment can be formulated into the
therapeutic
composition as neutralized pharmaceutically acceptable salt forms.
Pharmaceutically
acceptable salts include the acid addition salts (formed with the free amino
groups of the
polypeptide or antibody molecule) and which are formed with inorganic acids
such as,
for example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic,
3o tartaric, mandelic, and the like. Salts formed from the free carboxyl
groups can also be
derived from inorganic bases such as, for example, sodium, potassium,
ammonium,


CA 02340465 2001-02-21
WO 00/11168 25 PCT/US99/19052
calcium, or ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The therapeutic polypeptide-, analog- or active fragment-containing
compositions are conventionally administered intravenously, as by injection of
a unit
s dose, for example. The term "unit dose" when used in reference to a
therapeutic
composition of the present invention refers to physically discrete units
suitable as
unitary dosage for humans, each unit containing a predetermined quantity of
active
material calculated to produce the desired therapeutic effect in association
with the
required diluent; i.e., carrier, or vehicle.
to The compositions are administered in a manner compatible with the dosage
formulation, and in a therapeutically effective amount. The quantity to be
administered
depends on the subject to be treated, capacity of the subject's immune system
to utilize
the active ingredient, and degree of inhibition desired. Precise amounts of
active
ingredient required to be administered depend on the judgment of the
practitioner and
~ 5 are peculiar to each individual. However, suitable dosages may range from
about 0.1
to 20, preferably about 0.5 to about 10, and more preferably one to several,
milligrams
of active ingredient per kilogram body weight of individual per day and depend
on the
route of administration. Suitable regimes for initial administration and
booster shots are
also variable, but are typified by an initial administration followed by
repeated doses at
20 one or more hour intervals by a subsequent injection or other
administration.
Alternatively, continuous intravenous infusion sufficient to maintain
concentrations of
ten nanomolar to ten micromolar in the blood are contemplated.
As used herein, the term "synthetic amino acid" means an amino acid which is
chemically synthesized and is not one of the 20 amino acids naturally
occurring in nature.
25 As used herein, the term "biosynthetic amino acid" means an amino acid
found in nature
other than the 20 amino acids commonly described and understood in the art as
"natural
amino acids."
As used herein, amino acid residues are preferred to be in the "L" isomeric
form. However, residues in the "D" isomeric form can be substituted for any L-
amino
3o acid residue, as long as the desired functional property is retained by the
polypeptide.
NHz refers to the free amino group present at the amino terminus of a
polypeptide.
COOH refers to the free carboxy group present at the carboxy terminus of a


CA 02340465 2001-02-21
WO 00/11168 26 PCT/US99/19052
polypeptide. Abbreviations for amino acid residues are used in keeping with
standard
polypeptide nomenclature delineated in J. Biol. Chem., 243:3552-59 (1969).
It should be noted that all amino-acid residue sequences are represented
herein
by formulae whose left and right orientation is in the conventional direction
of amino-
s terminus to carboxy-terminus. Furthermore, it should be noted that a dash at
the
beginning or end of an amino acid residue sequence indicates a peptide bond to
a further
sequence of one or more amino-acid residues.
Amino acids with nonpolar R groups include: Alanine, Valine, Leucine,
Isoleucine, Proline, Phenylalanine, Tryptophan and Methionine. Amino acids
with
to uncharged polar R groups include: Glycine, Serine, Threonine, Cysteine,
Tyrosine,
Asparagine and Glutamine. Amino acids with charged polar R groups (negatively
charged at Ph 6.0) include: Aspartic acid and Glutamic acid. Basic amino acids
(positively charged at pH 6.0) include: Lysine, Arginine and Histidine (at pH
6.0).
Amino acids with phenyl groups include: Phenylalanine, Tryptophan and
Tyrosine.
is Particularly preferred substitutions are: Lys for Arg and vice versa such
that a positive
charge may be maintained; Glu for Asp and vice versa such that a negative
charge may
be maintained; Ser for Thr such that a tree -OH can be maintained; and Gln for
Asn
such that a free NHz can be maintained. Amino acids can be in the "D" or "L"
configuration. Use of peptidomimetics may involve the incorporation of a non-
amino
2o acid residue with non-amide linkages at a given position.
Amino acid substitutions may also be introduced to substitute an amino acid
with
a particularly preferable property. For example, a Cys may be introduced a
potential
site for disulfide bridges with another Cys. A His may be introduced as a
particularly
"catalytic" site (i.e., His can act as an acid or base and is the most common
amino acid
2s in biochemical catalysis). Pro may be introduced because of its
particularly planar
structure, which induces -turns in the protein's structure.
As used herein, "pM" means picomolar, "nM" means nanmolar, "uM, means
micromolar, "mM" means millimolar, "ul" or "ul" mean microliter, "ml" means
milliliter,
"1" means liter.
The following examples are presented in order to more fully illustrate the
preferred embodiments of the invention. They should in no way be construed,
however,


CA 02340465 2001-02-21
WO 00/11168 27 PCT/US99/19052
as limiting the broad scope of the invention. While the invention is described
and
illustrated herein by references to various specific material, procedures and
examples, it is
understood that the invention is not restricted to the particular material
combinations of
material, and procedures selected for that purpose. Numerous variations of
such details
can be implied as will be appreciated by those skilled in the art.
EXAMPI ES
EXAMPLE 1: Molecules specifically expressed in murine stem/progenitor
1 o cells
Protein tyrosine kinases and phosphatases expressed in murine hematopoietic
stem cells have been previously identified (Matthews, W., et al. 1991;
Matthews, W., et
al. 1991; Dosil, M., et al. 1996). These molecules play important roles in
hematopoiesis
and development (Dosil, M., et al. 1996; Mackarehtschian, K., et al. 1995;
Shalaby, F., et
al. 1995; Kabrun, N. , et al. 1997). The present invention contemplates an
even more
global approach in order to identify molecules specifically expressed in the
murine
stem/progenitor cell hierarchy. For most of these studies purified (AA4.1+Lin-
/loSca+ckit+) fetal liver cells were used. This population is approximately
1,000-fold
2o enriched for in vivo repopulating activity (LTRA) measured by competitive
repopulation
(LyS.I/Ly5.2 congenic system) (Moore, K.A., et al. 1997) . All myeloid and
lymphoid
lineages in primary and in secondary recipients are repopulated by these
cells. This
degree of enrichment is comparable to the current "state of the art." Other
primitive
members of the stem/progenitor cell hierarchy share this same cell surface
phenotype.
These include: ( 1 ) LTCIC or cobblestone area forming cells (Ploemacher,
R.E., et al.
1989; Ploemacher, R.E., et ai. 1991) , (2) CFU-blast progenitors , (3) HPP-CFC
progenitors (Lowry, P.A., et al. 1995) and (4) stromal-dependent B-lymphoid
progenitors
(Whitlock, C.A., and Miiller-Sieburg, C.E. 1990) . The AA4.1+Lin-/loSea-ckit+
subset is
depleted of LTRA but contains significant in vitro progenitor activity. In
contrast, no
3o stem/progenitor cell activity is found in the AA4.1 subset (Jordan, C.T.,
et al. 1990) .
Short-term (5-7 days) cytokine cultures of stem cells were used to generate
committed
progenitor populations at the expense of LTRA (Traycoff, C.M., et al. 1996;
Peters, S.O.,
et al. 1995; Knobel, K.M., et al. 1994; Yonemura, Y., et al. 1996). In
summary, several


CA 02340465 2001-02-21
WO 00/1116$ 28 PCTNS99/19052
cell populations were defined which represent the beginning, the middle and
the end
points of the hematopoietic hierarchy. This sets the stage for a comparative
analysis of
gene expression patterns. A goal of the present invention is to complement the
physical
and functional phenotypes of stem/progenitor cells with profiles of uniquely
expressed
genes. It was hypothesized that some of these gene products contribute to the
unique
biological properties of primitive stem/progenitor cells and therefore are
regulators of
self renewal, proliferation, commitment and other processes.
There are a number of ways to compare gene expression profiles which are
available to one of skill in the art. It is possible to do this by exhaustive
sequencing of
to representative cDNA collections obtained from stem cell and mature cell
sources
followed by "electronic subtraction". This approach has several drawbacks.
Most
importantly, the number of sequences which must be obtained is prohibitive.
For a
homogeneously pure population this number is on the order of 50,000 (based on
approximately 10-20,000 expressed genes in an average cell type and a
statistical
calculation). In practice, even the most purified stem cell population is
heterogeneous.
Stem cell enrichment values are only meaningful in relation to an unenriched
standard
and cannot be converted into an absolute stem cell number. It has been
documented that
cell populations with the same cell-surface phenotypes can differ in
biological activity .
The unique properties of stem cells also suggest caution when extrapolating
from
2o expressed gene numbers in other cells. In short, the extent of sequencing
necessary to
ensure complete coverage of gene expression in stem cells is not possible to
estimate.
Normalization procedures (Uchida, N., et ai. 1995; Patanjali, S.R., et al.
1991; Soares,
M.B., et al. 1994) designed to "equalize" the mRNA abundance classes are not
advisable
because they obliterate potentially important quantitative expression
differences.
Additionally, a high-throughput sequencing effort is not applicable to
numerous libraries.
Comparisons of gene expression in diverse sources of stem cells will provide
valuable
information. An elegant technique, Serial Analysis of Gene Expression (SAGE)
permits
the rapid acquisition of thousands of DNA sequence "tags" (Zhang, L., et al.
1997) . This
technology was not considered herein because the size of each sequence "tag"
is very
3o small ( 10 base pair, bp). Therefore, SAGE is only informative in two
extremes; exact
nucleotide matches or no matches to sequences in the databases. This limits
database
comparison to the same species from which the "tags" originate. A key
component of the
strategy presented herein relies on broad bioinformatic database comparisons.
In


CA 02340465 2001-02-21
WO 00/11168 29 PCT/US99/19052
addition, even with a specific "tag" one still needs to obtain a full-length
cDNA clone for
functional studies. Analysis of gene expression can be done in single
progenitor cells
after these form a colony "start" (Brady, G., et al. 1990; Brady, G., and
Iscove, N.N.
1993). Replating of the sibling cells in a "start" colony allows the
approximation of the
lineage potential present in the starting cell. This technique suffers from
several
drawbacks. First, it is limited to 3', non-coding ends, thus preventing
protein database
comparisons. Second, the technique relies on cell growth, thus it is not
suitable for
analysis of quiescent cells. Moreover, approaches to allow colony-formation by
true stem
cells with a retention of primitive properties are in their infancy (Ball,
T.C., et al. 1995;
to Trevisan, M., et al. 1996). Third, the technique does not take into account
stochastic
models of stem cell behavior (Ogawa, M. 1993). To accurately reveal physical
and
functional properties of stem cells it is wise to analyze populations where
stochastic
differences would average out. Single-cell derived cDNA populations could
however,
provide valuable "adjunct" material for more refined gene expression screens.
There are
many techniques to physically identify differentially expressed genes. For
these and
other reasons, most notably the technical and economic ease with which
physical pre-
enrichments of cDNA libraries can be achieved, the studies described herein
began with
cDNA libraries which are highly enriched in differentially expressed
sequences. The
integration of individual techniques served to overcome the inherent
limitations of each
2o technique. Three strategies were successfully employed: ( 1 ) Differential
Display (DD)
(Liang, P., et al. 1994; Bauer, D., et al. 1993), (2) Representational
Difference Analysis
(RDA) (Braun, B.S., et al. 1995; Hubank, M., and Schatz, D.G. 1994;
Diatchenko, L., et
al. 1996) and (3) standard subtractive hybridization (Li, W.-B., et al. 1994;
Harrison,
S.M., et al. 1995) . The latter underlies the present invention. A key feature
is that the
differentially expressed cDNAs have a high probability of being full-length.
This
facilitates a rapid transition to functional studies. The two former
techniques were
utilized because of "visual" nature (DD) and the ability to generate
representative,
differentially expressed probe populations (RDA) in a rapid manner.
Murine stem cell eene expression rofiles. As a first step, a series of high
quality,
3o representative cDNA libraries were generated. The cDNA populations were
directionally
cloned into the pSport-1 or pSport-2 plasmids (BRL-Gibco). The most important
libraries originate from purified stem cells. In one case, enough AA4.1+Lin
/IoSca+ckit+cells were purified to allow construction of a non-based library
using


CA 02340465 2001-02-21
WO 00/11168 30 PCT/US99/19052
standard methodologies. This library contains 4x106 independent recombinants
(average cDNA insert size of 1-2 kilobases, kb). A second library was
constructed using
a new PCR-based technology called cap-finder (Clontech) designed to yield full-
length
cDNA copies. cDNAs ranging from 1 to 4 kb were commonly attained using this
technique. For cap-finder procedures the purified cells were processed into
DNAse-I
digested, poly-A+ mRNA according to microscale procedures routinely in use.
The
synthesis of cDNA was done with an aliquot of mRNA corresponding to
approximately
20,000 cell equivalents. It has not been necessary to use less material,
therefore this is
not a lower Limit. A Not I restriction site was included in the 3' reverse
transcriptase
o primer (cap-finder, version 2) to facilitate directional cloning. An aliquot
of the cDNA
was amplified for varying PCR cycle numbers, and analyzed by Southern blots
(pseudo-
Northerns) for the presence of full-length copies of b-actin, GAPDH, CD 18,
flk2/flt3,
cdk4, CD34 and other mRNAs. Optimal cycle numbers were used to amplify the
remaining cDNA. The cDNA was cloned into the pSport-1 plasmid. The AA4.1+Lin-
~ 5 /loSca+ckit+ cap-finder library contains ~3 x 106 independent
recombinants. Single-pass
sequence analysis of random clones from this library indicated that 95% were
full-length
(based on sequences with an exact match in Genbank). Much more extensive
sequencing
of numerous clones from subtracted libraries has confirmed this. Other
libraries
constructed in similar ways include two libraries from AA4.1 cells, and two
libraries from
2o AA4.1+Lin-/IoSca-ckit+ cells. Finally, a library was constructed from
AA4.1+Lin-
/loSca+ckit+ cells cultured for 5-7 days in a differentiation promoting
cytokine cocktail
(IL3, IL6, KL). Competitive repopulation and in vitro progenitor assays
confirmed a
complete loss of LTRA with a significant retention of progenitor cells. All of
the cDNA
libraries are large (> 2x106 independent recombinants) and of high quality {1-
2 kb
25 average insert size). In summary, the most primitive, intermediate and most
mature
members of the hematopoietic hierarchy were "converted" into representative
panels of
expressed genes.
Subtractiv-~ Hybri ization. These cDNA libraries were used in subtractive
hybridizations to enrich for differentially expressed genes. Target libraries
from
3o AA4.1+Lin-/loSca+ckit+ cells were subtracted with an AA4.1 driver cDNA
library. This
yields a population of cDNA clones which is enriched in sequences expressed in
primitive stem/progenitor cells but not in mature cell types. In practice, a
single-stranded
target library is hybridized to an excess of in vitro synthesized biotinylated
RNA from the


CA 02340465 2001-02-21
WO 00/11168 31 PCT/US99/19052
driver library as described . The opposite orientations of the cloning sites
in pSport-1 and
2 insure target and driver complementarity. The target library was
enzymatically
converted to a single-stranded form using gene II protein and exonuclease III
(Gene-
Trapper protocols, BRL-Gibco). Previously, this was done by infection with M13
helper
phage often resulting in a bias for small cDNA inserts. Here, driver/target
combinations
were subtracted two times in order to facilitate the removal of commonly
expressed
sequences (verified by elimination of "housekeeping" genes such as b-actin and
GAPDH). Concomitant enrichment of known, differentially expressed genes is
also
verified. Generally flk2/flt3 and CD34 probes were used. Both are expressed in
the
to AA4.1+Lin-/loSca+ckit+ subpopulation but not in the AA4.1 population.
Following
subtraction, the relative number of clones is reduced by up to 200-fold. In
some cases
(subtractions with AA4.1 material) the number of clones which "survive" the
subtraction
is on the order of 10-20,000. Because individual sequences may be represented
more
than one time, this does not necessarily imply that there are 10-20,000
differentially
is expressed genes. The exact number of unique sequences (complexity) in the
pool of
subtracted clones must be determined. A more thorough discussion of complexity
is
found in a subsequent section. These subtracted libraries should be enriched
for
sequences expressed in the primitive portion of the stem/progenitor cell
hierarchy; that is
in stem cells and/or in primitive clonogenic progenitor cells. Two other
subtracted
20 libraries, potentially enriched for sequences expressed in the most
primitive stem cell but
not in clonogenic progenitors were derived by subtracting the AA4.1+Lin-
/loSca+ckit+
libraries with material from the closely related AA4.1+Lin-/IoSca-ckit+
subpopulation.
Each subtracted library is arrayed at high density onto nylon membranes. Each
clone in the array has a unique address in microtiter plates. A density of 20-
30,000 clones
25 on a 22 x 22 cm. membrane is practical. Because of the inherent "noise" in
any
subtraction scheme, a positive selection criterion may be imposed in order to
focus on
true differentially expressed sequences. Details are presented in a subsequent
section. To
analyze the subtracted libraries, a high throughput sequencing effort was
employed using
three libraries. These are: 1) two AA4.1+Lin-/loSca+ckit+ cell libraries
(standard and
3o cap-finder) subtracted extensively with AA4.1 cell material and 2) a
standard
AA4.1+Lin-/loSca+ckit+ library subtracted with AA4.1+Lin-/IoSca-ckit+
material. An
average of 400 by of 5' sequence was obtained from about 1000 clones. To
facilitate a
rational handling of sequence information and to focus attention on a small
number of


CA 02340465 2001-02-21
WO 00/11168 32 PCT/US99/19052
clones for functional analysis a "flow of information" strategy was devised.
(See, Figure
1).
A major component of this strategy is bioinformatics. This can be global
(comparisons with outside databases) and local ("in-house" analyses within an
individual
library or comparison of several libraries). A relevant example of the latter
is comparison
of genes from murine stem cells with genes derived from their human
counterparts.
Global bioinformatic analysis provides much information. First, it establishes
if a given
sequence corresponds identically or closely to an already identified gene in
the mouse,
human or other mammalian species. Such homologies can be detected at the
nucleotide
io level. This can provide evidence of hematopoietic expression for a
previously described
gene. Second, where the homologies are statistically significant but not
identical, novel
members of gene/protein families can be identified. Third, a wider net can be
cast over
the databases by using conceptual translation of a sequence in the homology
comparisons.
The Examples provided herein illustrate the power of this approach in
revealing
similarities to proteins from invertebrate organisms such as Drosophila, C.
elegans, and
even yeast. In many cases these proteins have functions which have been
uncovered by
the analysis of mutants. Based on protein homologies "virtual links" are drawn
between
developmental regulation in invertebrates (such as in germ-line development)
and in
hematopoietic stem cells. The Notch/Notch ligand pathway first defined in
invertebrate
2o cell-fate determination and recently implicated in hematopoietic regulation
is a good
example .
A number of other putative proteins which share homology with key Drosophila
proteins have been identified. Bioinformatics facilitates the recognition of
peptide motifs
such as EGF-like repeats, Ig-like domains or Zn-finger modules. Fourth,
because the
databases are annotated, predicted protein sequences can be assigned to
cellular processes
such as signal transduction pathways or apoptosis. It is also possible to
categorize clones
according to potential involvement in other mammalian stem cell systems such
as the
intestine. Fifth, it is feasible to perform virtual expression studies and to
construct
overlapping EST contigs which can yield virtual full-length cDNAs.
3o The following discussion summarizes the general murine findings and
highlights a
panel of "interesting" subtracted clones. A number of full-length sequences
have been
determined. Bioinformatic analysis summaries on a collection of 863 clones
derived


CA 02340465 2001-02-21
WO 00/11168 33 PCT/US99/19052
from AA4.1+Lin-/loSca+ckit+ libraries subtracted with AA4.1 RNA are shown in
Table
1.
The present invention encompasses numerous bioinformatic search and
comparison parameters. This is the first analysis of its kind in the
hematopoietic system
and several important points emerge. First there is a high proportion (~SO%)
of novel
sequences. Sixteen percent do not show homologies in any EST database queried.
A
preliminary analysis of a small sequence set from an AA4.1+Lin-/loSca+ckit+
library
depleted of sequences in common with the AA4.1+Lin-/loSca-clot+subset has
indicated
an even higher percentage of novel genes. These are higher percentages than
would
to emerge from a random analysis of an unsubtracted library. This was directly
addressed by
performing these analyses on a similar number of sequences selected at random
from one
of the mouse EST databases. Second, among the ~50% of the clones which are
significantly homologous to previously identified genes or proteins, the
proportion of
homologies to "housekeeping" genes is low. This underscores the effectiveness
of the
subtraction strategy. Third, an internal analysis of the data base has
revealed few
redundancies. This illustrates the degree of gene expression diversity between
the two
endpoints of the mouse hematopoietic hierarchy; thus supporting the starting
hypothesis.
A recent report has suggested that many sequences in mature blood cells are
expressed in
primitive, non-committed cells (Hu, M., et al. 1997). The data indicate that
many of these
2o are removed by the subtraction; thus uncovering a large, previously not
described set of
genes. Fourth the sequences with homologies to known genes or proteins can be
subdivided according to protein families and putative function. Interestingly,
a large
percentage (32%) fall into the signaling protein category. Examples of these
are
described herein below.
As part of a more sophisticated bioinformatics approach, automation of
database
searches, information cross-referencing, and annotation was employed. An
illustrative
example is automated weekly database queries with the sequence set. New,
previously-
unidentified homologies were automatically noted and reported. In the mouse
studies
several genes were encountered whose expression would be predicted to differ
between
3o the AA4.1+Lin-/loSca+ckit+ and the AA4.1- populations. Both murine CD34 and
flk2/flt3 were identified in the sequenced population. This provides a good
internal
control for the screening strategy. As shown in Table 2, a "short list" of
identified


CA 02340465 2001-02-21
WO 00/11168 34 PC'T/US99/19052
molecules was generated based on bioinformatics and in many cases, expression-
specificity verification. The features of some of the molecules merit
discussion,
The SA49P1 clone is homologous to sex comb on midleg; a member of the
Drosophila Polycomb group of zinc-finger transcriptional repressors
(Bornemann, D., et
al. 1996). Polycomb proteins are regulators of homeobox (HOX) genes and
maintain the
developmental stability of transcriptional states (Simon, J. 1995) . There is
currently
great interest in HOX gene function in the biology of hematopoietic stem cells
(Sauvageau, G., et al. 1995; Lawrence, H.J., et al. 1996) . The C4-80 gene is
homologous
to the Drosophila cornichon gene (Roth, S., et al. 1995) . Cornichon is
required during
to oogenesis for the induction of follicle cells, which provide the
environment that supports
oocyte development. Cornichon is a component of the Drosophila EGFR signaling
pathway (Neuman-Silverberg, F.S., and T. Schupback 1996). The LL2-12 gene is
similar
to the Drosophila neurogenesis gene brainiac. Mutants defective in this
extracellular gene
product show neural hyperplasia. Brainiac also plays a role in oogenesis
(Goode, S., et al.
t5 1992; Goode, S., et al. 1996) . Mosaic experiments show that brainiac is
required in the
developing oocyte. Brainiac involvement in the Drosophila EGFR pathway has
been
suggested. The LL2-35 gene is similar to the Drosophila germ cell-less gene
(Jongens,
T.A., et al. 1992) . The product of this gene is required for the
specification of the
Drosophila germ line. The ectopic expression of germ cell-less causes somatic
cells to
zo adopt the characteristics of pole cells (destined for the germ line). The
homologies of the
above clones to Drosophila genes were identified at the predicted amino acid
level. This
underscores the utility of the bioinformatic approach. In all four cases the
cDNAs
represent novel unpublished murine genes. Clearly, the involvement of three of
the
Drosophila genes in the ultimate stem cell system, the germline, coupled with
the
25 identification of homologous genes expressed in mouse hematopoietic stem
/progenitor
cells gives food for thought. Four other cDNAs identified are homologous to
the
Drosophila genes dishevelled and smoothened (mouse homologs are already
described) as
well as to ketch and discs large (not previously described in the mouse).
DD116 was
originally isolated in a DD comparison of AA4.1+ vs. AA4.1 cells. A cDNA clone
3o identified with the DD116 probe was sequenced; the predicted protein is
homologous but
not identical to the murine beige gene product. The beige mutation in the
mouse causes
bleeding, immune system disorders and a coat-color phenotype (Perou, C.M., et
al. 1996;
Fukai, K., et al. 1996; Barbosa, M.D., et al. 1996). Murine beige is thought
to be the


CA 02340465 2001-02-21
WO 00/11168 35 PCT/US99/19052
homologue of a gene responsible for the Chediak-Higashi syndrome in human.
This
novel gene is likely to be the second member in the beige family; it is
designated herein
as "taupe". Two of the cDNAs in Table 2 were identified in a DD comparison of
AA4.1+Lin-/IoSca+ckit+ cells and cytokine differentiated, cultured cells. One
focus was
molecules whose expression disappeared with the loss of stem cell activity in
culture.
Cyt28 and Cytl9 fulfilled this criterion and were used to isolate full length
clones which
have been completely sequenced. Cyt28 encodes a novel, seven-transmembrane
domain
receptor in the same family as mouse F4/80 and human CD97 (secretin receptor
superfamily) (Hamann, J., et al. 1995; Baud, W., et al. 1995) . Cyt 19 is a
novel putative
to methyltransferase. In both of these cases protein family assignations were
made after
extensive bioinformatic analyses. At least in the case of Cyt28, an antibody
will be a
useful reagent because it may provide a means to further subdivide the
stem/progenitor
cell population. Such antibodies are provided by the present invention. The
cDNA SA61
is similar to a newly discovered molecule called p62dok. p62dok is a tyrosine
~ 5 phosphorylated protein which binds to rasGAP and is likely to be a common
target for
numerous tyrosine kinases including Abl and ckit (Carpino, N., et al. 1997;
Yamanashi,
Y., 1997) . Interestingly, p62dok is also constitutively phosphorylated in
hematopoietic
progenitor cells from chronic phase CML patients (Carpino, N,, et al. 1997) .
The
predicted protein encoded by SA61 appears to be a second member of this
protein class.
20 Other representative molecules are listed in Table 2. Two cDNAs were
identified as
homologous to putative apoptosis regulators (SBSA56 and LLS-68, two cDNAs
homologous to genes translocated in leukemias (LLS-03 and B2-67), several
homologies
to putative chromatin proteins (C4-23, C3-25 and LL2-89) and a LIM-domain
encoding
cDNA (LL5-96). Most interestingly, three eDNAs (B3-77, C2-48, and LL2-76) are
25 homologous to genes expressed in intestinal crypts or during intestinal
development.
Isolated cDNAs were hybridized to slot blots representing globally amplified
cDNAs from pools of individual progenitor and mature cells. Because the
developmental
potentials of these cells have been measured (by replating of siblings) it is
possible to
graphically represent the expression of a gene in various stages of the
hierarchy. Three
3o examples are shown in Figures 2A-2C. The Smc-34 cDNA is a completely novel
sequence with a predicted leucine zipper and several potential membrane
spanning
domains. These examples underscore the value of "interfacing" the above-
described
approach with a single cell approach. Thus, it was demonstrated that a given
gene is


CA 02340465 2001-02-21
WO 00!11168 36 PCTNS99/19052
expressed in at least multipotential progenitor cells and other members of the
progenitor
cell hierarchy.
~fuman stem cell gene expression profiles. It is also a goal of the present
invention to identify genes expressed specifically in human stem cells. It is
useful to use
a mufti-species (mouse and human) approach to define stem/progenitor cell-
specific gene
profiles. One goal is to integrate mouse and human information. However,
rather than
proceeding directly to human homologs of the mouse genes already identified,
an
independent human effort was initiated, The rationale for this is mufti-
faceted. First, it is
possible that some aspects of human and mouse stem cell biology are regulated
in
to different ways. Although it is likely that most regulatory pathways will be
conserved, it
must be kept in mind that many properties ascribed to the most primitive stem
cell
population have been rigorously proven only in the mouse. Clearly a human
lymphoid-
myeloid stem/progenitor cell exists. However, the exact degree of
proliferative capacity
(the ability to give rise to oligoclonal hematopoiesis) as well as the exact
spectrum of
differentiation potentials of human stem cells have not yet been accurately
measured.
Several potential differences between mouse and human have already been
mentioned.
One additional indicator of differences may be the difficulty of gene-transfer
into stem
cells from large animals (Larochelle, A., et al. 1997). This is not likely to
result solely
from the quiescent status of the most primitive cells. Second, while it is
usually possible
2o to find human homologs for individual mouse genes by manipulating
hybridization
stringency, such conditions will vary for different genes. Therefore, to find
human
homologs for a large pool of mouse genes (i.e. 100) may be more labor
intensive and
costly than to independently determine a sequence profile of differentially
expressed
genes from human stem/progenitor cells. Relationships to the murine panel can
then be
determined electronically where it is easy to manipulate comparative
parameters.
Clearly, for some individual mouse genes it will be of great importance to
physically
identify human homologs. In some cases it may be possible to use existing
human EST
databases to quickly obtain the sequence of a human homolog. Third, the
availability of a
large panel of human sequences specifically expressed in stem/progenitor cells
lends
itself to the application of various chip and array technologies. Such
technologies will be
instrumental in identifying which subsets of human stem cell specific genes
are up or
down-regulated in the highly clonogenic stem cells from diseases such as Acute
Myelogenous Leukemia (AML) and other leukemias.- Highly purified human stem


CA 02340465 2001-02-21
WO 00/11168 37 PCT/US99/19052
cells for cDNA libraries were obtained from normal donor BM. CD34+ cells were
obtained from the mononuclear fraction using an immuno-magnetic affinity
device
(Miltenyi MACS column) and stained with a mixture of monoclonal antibodies
(mAbs)
designed to identify both primitive and mature cell surface markers. To
identify antigens
on mature cells a lineage (Lin) cocktail of FITC conjugated mAbs was used .
These
mAbs were directed against: CD3, CDllb, CD15, CD19, CD20, glycophorin A, and
CD71 (CD3, CD15, CD19 and CD20 from Becton Dickinson, CDllb and glycophorin A
from Coulter, and CD71 from Ortho Diagnostics). To identify primitive cells
mAbs were
used which were directed against CD34 (biotin conjugated and detected via a
1o streptavidin-allophycocyanin reagent; Coulter) and either CD38
(phycoerythrin
conjugated, Becton Dickinson) or CD90 (Thyl) (phycoerythrin conjugated;
Pharmingen).
The stained cells were analyzed using a dual laser Becton Dickinson FACStar
Plus flow
cytometer. Cells of the desired phenotype were sorted into siliconized tubes.
To obtain
material from cultured cells, CD34+ enriched populations (isolated as
described above),
t5 were first cultured in serum-free medium (IMDM, 5 mg/ml low density
lipoprotein, 2
mM glutamine, human serum albumin, insulin, and transfernn, ) supplemented
with IL-3
(Sng/ml), KL (25ng/ml), and FL (25ng/ml). After 1-4 days of culture, the cells
were
purified as described above. Human libraries were constructed using the cap-
finder
technology, version 2. Amplified cDNAs originating from several independent BM
2o donors were pooled. Representative cDNA libraries have been constructed
from the
following sources: ( 1 ) BM CD34+Lin- cells (7.1 X/ 05 independent
recombinants), (2)
BM CD34+Lin-CD38+ cells (1.9X106 independent recombinants), (3) BM CD34- cells
(1.6X106 independent recombinants), (4) CB CD34+ cells (4.3X105 independent
recombinants}, and (5) CB CD34- cells (2.9X105 independent recombinants). The
25 average cDNA insert size in all libraries is 1-2 kb. According to the
comparative
biological properties of the source material used for the mouse and human cDNA
libraries, the following parallels can be drawn: (1) mouse AA4.1+Lin-
/loSca+ckit+
human CD34+Lin-, (2) mouse AA4.1+Lin-/loSca-ckit+ = human CD34+Lin-CD38+ and
(3) mouse AA4.1 - human CD34-. Thus, in both species, collections of clones
3o representing the beginning, the middle and the end-points of the
hematopoietic hierarchy
have been generated. Using procedures described above, the human libraries
were
subtracted and arrayed 3,000 clones from two libraries: (1) BM CD34+Lin-
subtracted


CA 02340465 2001-02-21
WO 00/11168 38 PCT/US99/19052
with BM CD34+Lin-CD38+ material (refereed to as: 38-)38+) and (2) BM CD34+Lin-
subtracted with BM CD34- material (referred to as: 38-). Before initiating a
sequencing
effort pilot studies were performed to improve the resolution of the screen.
Specifically,
in order to develop techniques which would eliminate as much non-specific
"noise" as
possible from the libraries. RDA (PCR-Select, Clontech) were used . There are
several
salient features of RDA. First, it is rapid (2-3 days) and applicable to small
cell numbers
(ie. several thousand). It does not require the generation of cloned cDNA
libraries
therefore, numerous comparisons can be made simultaneously. Second, it yields
short
cDNA fragments (generated by 4-cutter restriction enzymes) which are
representative of
to differentially expressed mRNAs. Third, it is based on the suppression PCR
technique
which prevents overamplification of abundant messages (Diatchenko, L., et al.
1996) . In
addition, sequences expressed in both populations in an RDA comparison do not
amplify
exponentially. Differentially expressed populations of cDNA fragments are not
obtained
by physical enrichment but rather by selective PCR amplification. Fourth and
most
importantly, it simultaneously yields two populations each representing
differentially
expressed genes in one of the two starting samples. This bi-directionality is
valuable
because as discussed below it can simultaneously reveal up and down regulated
genes.
RDA comparisons were made between CD34+Lin- and CD34+Lin-CD38+ and between
CD34+Lin- and CD34- cDNAs. The latter comparison was verified by
hybridization. As
2o shown in Figure 3A (right panel), the control, non-subtracted RDA cDNA
population (38
contains b-actin sequences which are missing in the two subtracted RDA
populations,
38- and 38-)38+. A differentially expressed gene (HDD-2, described below) is
enriched
in the 38- RDA population and at least retained in the 38-)38+ RDA population
(Figure
3A, left panel). Two, bi-directional, RDA cDNA populations (38- and the
converse 34
)38) were used to probe (See Figure 3) duplicate arrays of a subtracted 38-
library (Figure
3B). The correct RDA probe hybridizes to considerably more clones than the
incorrect
probe (compare the greater numbers of spots in the left as compared to the
right panel).
Hybridization signals for each clone are doublets due to the arraying
technique.
Hybridization signals with the incorrect probe (right panel} suggest that
further
3o improvements to this strategy are worth pursuing. Because the subtractions
are based on
different technologies, it was reasoned that clones which "survived" the
library
subtraction and hybridized preferentially to the "correct" RDA probe would be
more
likely to represent true differentially expressed genes. Preliminary data from
the mouse


CA 02340465 2001-02-21
WO 00/11168 39 PCTNS99119052
studies had already suggested that this was indeed the case. The arrayed
clones were also
hybridized to probe populations from the entire starting non-subtracted
libraries in order
to eliminate residual cDNA clones corresponding to abundant mRNA species.
In one analysis, a total of 77 clones (49 from the 38- subtraction and 28 from
the
38-)38+ subtraction) which "obeyed" the RDA criteria were sequenced from the
5' end.
In order to verify expression specificity, a series of pseudo-Northern blot
analyses were
performed. cDNA populations from independent purified sources of material was
amplified. This is of particular importance with an outbred species such as
humans. In
these experiments additional material from CD34+Lin-CD90+ (Thyl+) (three
separate
l0 purifications) and their CD90- (Thyl-) counterpart subpopulations was also
included.
Cytokine-cultured samples were also included. These were CD34+Lin- cells and
their
CD38+ counterparts purified after 1, 2 and 4 days of culture. The collective
panel of
cDNAs also includes four independently purified CD34+Lin- populations and
their
CD34+Lin-CD38+ counterparts. All of these ampiified cDNAs have been arranged
on
numerous pseudo-Northern blots. In short, this allowed us to evaluate the
expression of a
given "sequence tag" in stem cell populations isolated by two different
criteria, from a
number of independent donors and after cytokine culture. Several interesting
genes
emerged from the sequence data set. Individual clones obtained from the -38-
)38+
subtraction are designated 38 letterlnumber (i.e., 38A1) while those from the
38-)34+
subtraction are designated 34 letter/number (i.e., 34A1). One clone (38B5) was
identified
and determined to be human flk2/flt3. A second interesting clone (34B4) is
closely
related to a gene encoding TINUIt (Figure 4A). Clone 34B4 may be a novel
variant of
TINUR due to a 25 amino acid, in-frame deletion. TINUR was identified as an
orphan
member of the steroid receptor superfamily (NGFI-B/nur77 subfamily)(Okabe, T.,
et al.
1995). TINUR has also been implicated in apoptosis. An additional clone (34F4
) is
highly homologous to DAP-kinase. This protein is a serine-threonine kinase
which has
been implicated in cytokine (IFN- -induced apoptosis (Deiss, L., et al. 1995)
. Clone
34F4 (DAP-Kinase) and 34B4 (TINUR) both exhibit a stem-cell restricted
expression
pattern. Clearly, the identification of two genes whose products are
implicated in
3o apoptosis and whose expression is largely restricted to human stem cells is
of interest. In
addition, a cDNA (34A5) was identified which is closely related to the MLF1
gene which
is a translocation partner in t(3;5)(q25.1;q34)(Yoneda-Kato, N., et al. 1996)
. This
translocation is associated with Myelodysplastic Syndrome (a stem cell
disease) and


CA 02340465 2001-02-21
WO 00/11168 40 PCT/US99/19052
AML. The sequence homologies and restricted expression pattern of 34A5 is
shown in
Figures 4B and 4C. In Figures 4C and 4D (and also SA, SB, and 6B) there are
twenty-
one samples of capfinder-amplified cDNA from various hematopoietic
populations.
From left to right these are: four CD34+Lin- populations, three CD34+Lin-CD90+
populations, two CD34- populations, four CD34+Lin-CD38+ (obtained from the
same
BMs as the CD38- samples in lanes 1-4), two CD34+Lin- samples (obtained from
the
same BMs as the CD90+ samples in lanes 5 and 6), three CD34+Lin- populations
obtained after 1, 2 or 4 days of culture and finally their three CD34+Lin-
CD38+
counterparts. A recent study shows that, at least with some types of AML, the
disease
to can be transferred into NODSCID mice only by the leukemic CD34+CD38-
subpopulation (Bonnet, D., and Dick, J.E. 1997) . These demonstrate MLFI
expression
in normal stem cells. As shown in Figure 4D, nucleophosmin (NPM), the partner
in this
translocation was also identified. This is an example of non-specifically
expressed
"noise" in the screens. The 3862 cDNA is closely homologous to the LTG9/MLLT3
t5 gene located on 9q22 and involved in t(9;11) leukemia (Iida, S., et al.
1993) (Figure 4E).
A degree of stem cell expression specificity has also been observed. All of
the above
cDNA clones are likely to be identical to the homologous, previously
identified genes.
However, the suggested involvement of DAP-kinase and TINLTR in apoptosis
necessitates
their inclusion in any comprehensive consideration of stem cell apoptotic
pathways.
2o Similarly, the expression of two genes associated with myeloid leukemias
bears on any
speculation regarding the primary transformation target cell as well as the
origins of the
ultimate clinical phenotype in these and other leukemic disorders.
Other known genes to were found that exhibit identity or very close homology
including: (I) GOS3, a fos-related gene (Heximer, S., et al. 1996) (Figure 5A)
and (2)
25 HLA-DR (Figure 5B). GOS3 shows a specific expression pattern, while HLA-DR
expression appears to be more variable. The expression status of Class II MHC
on the
most primitive human BM stem cell population is not entirely clear. It has
been
suggested to be present in a primitive, multipotent progenitor population but
not in the
most primitive stem cells (Sutherland, H.S., et al. 1989; Verfaillie, C., et
al. 1990) . If
3o true, this may suggest an additional negative selection parameter for
future experiments
designed to subdivide the stemlprogenitor cell hierarchy. Of the first 75
sequences, 22
have no homologies in the databases or homologies only to ESTs. Expression
analyses
on these clones are in progress. One gene of particular interest is called HDD-
2 as well as


CA 02340465 2001-02-21
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34B5, 34E1 and 38A11. The three latter designations reflect its isolation from
both the
38- as well as the 38-)38+ subtracted libraries. The designation HDD-2
reflects its
independent isolation in a limited DD "first look" at molecular differences in
the purified
cell populations. The likely full-length sequence of HDD-2 is 500 bp. It
contains a
short open reading frame of 89 amino acids (SEQ.ID.No.: 7i). The predicted
peptide
sequence is shown in Figure 6A. The 3' cDNA sequence contains a poly-A tail
preceded
by the canonical AATAAA poly-adenylation signal. Neither the nucleotide nor
the
predicted protein sequences of HDD-2 show homologies in any known gene or EST
database. The expression profile of HDD-2 shown in Figure 6B, demonstrates
that it is
to stem cell restricted. Also shown below (Figure 6C) is HDD-2 hybridization
to a dot blot
with numerous human pA+ mRNA samples (Clontech). HDD-2 hybridization is only
visible in kidney (the other "spots" are background). It was confirmed that
HDD-2
corresponds to a single-copy human gene by genomic Southern blot (Figure bD).
In
summary, the results of this very low throughput human sequencing effort
substantiate
the overall approach; that judicious pre-enrichments and selections will
result in rapid
identification of biologically interesting and often novel genes. Most
importantly, these
studies firmly establish the existence of genes whose expression correlates
with the most
primitive stem cell phenotype.--
Other data were generated using (1) a murine stromal cell line to support
enriched
2o human stem/progenitor cells, and (2) tetracycline regulatable retroviral
expression
vectors. The stromal cell line AFT024 is efficient in long-term, in vitro
maintenance of
LTRA in purified murine fetal liver and adult bone marrow populations
(Terstappen,
L.W.M.M., et al. 1991) . Additional data demonstrated highly-efficient
retroviral-
mediated gene-transfer into murine LTRA as well as into primitive in vitro
progenitors
during the AFT024 cocultures. Further, it has been shown that AFT024 is very
effective
in supporting ELTCIC. Specifically, the CD34+CD38- immunophenotype as well as
the
functional capacity of these cells is maintained. The latter was measured in
limiting-
dilution replating experiments. Moreover, it has been shown that limiting
numbers of
human CD34+Lin- cells can give rise to both B cells and NK-cells when cultured
on
3o AFT024. However, the supportive activities of AFT024 on mouse and human
stem/progenitor cells have been indistinguishable. In addition, more than 500
sequences
have been analyzed from an AFT024-specific subtracted cDNA library. A number
of
candidate stem cell regulators have been identified. Three of these are dlk
(Moore, K.A.,


CA 02340465 2001-02-21
WO 00/11168 42 PCT/US99/19052
et al. 1997), a novel BMP/TGF-b superfamily member and a novel selectin-
related
molecule. The present invention contemplates identifying human stem/progenitor
cell
receptors and/or ligands for the AFT024 specific proteins.
In order to facilitate functional studies of stem cell gene products several
retroviral
s gene-transfer vector systems were constructed and characterized. All of
these employ the
293T cell retroviral packaging system (Kinsella, T., and Nolan, G. 1996). High
titers of
virus can be produced transiently without the time and labor consuming effort
required
for stable producer cell lines. Large cDNA populations can also be converted
into virus
populations (Kitamura. T., et al. 1995). It is preferable to have inducible
(or repressible)
vectors which are also selectable. Also, the single-transcription unit
tetracycline (tet)
repressible vector was modified (Hofmann, A., et al. 1996). This vector
includes an
enhancer/promoter deletion in the 3' LTR. The tet system is currently one of
the best
inducible expression systems available; regulation over a several hundred-fold
range of
expression is observed (Shokett, P.E., and Schatz, D.G. 1996) . Cloning sites
were
15 introduced in order to insert cDNAs in a sense or antisense orientation. A
green
fluorescent protein (GFP) marker was also included in these vectors driven by
a
thymidine-kinase promoter (TK-GFP) (Yang, T., et al. 1996; Cheng, L., and
Kain, S.
1996). The cDNA fragment of interest is under tet regulation while GFP is
constitutively
expressed in transduced cells. In order to confirm this, NIH3T3 cells are
infected with a
20 LacZ virus and GFP~ cells sorted. The GFP+ cells express LacZ in the
absence of tet,
while LacZ expression is undetectable in most cells after the addition of tet.
The titer of
this model construct is approximately l04/ml. which is suitable for tissue
culture studies.
'table 1. Known genes (or extensive as homology):
436 (50..~%)


Grouped by function


by nucleotide: 407 Signaling/receptors: 133


by amino acid only: 29 Translational/post-: 62


Structural: 49


to mouse: 230 Transcriptional/post-: . 45


to human/other: 206 Cell fate: 35


Other. 87


Unknown function: 25


Novel genes: 427 (49.5%)
Homologous only to expressed sequence tags: 288 (33.3%)
No homology to any known nt or as sequences: I 39 ( I6.2%)


CA 02340465 2001-02-21
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43
Table 2.
~ueative familr Clone hlethod Cells compareddotes


7-transctumbnne Cyt-28 difL SC' fresh vs. secretin R supetfamiiy
receptor display 7d-cultured


MethyltransferaseCyt-19 dill. SC fresh vs.
display Td-cultured


C3-54 subtraction5C vs. AAd'


Aspartyl proteaseSAT subtractionSC vs. AAt'
r


Signal tnnsductionSA6l subtractionSC vs. AA4' dok fancily member
molecules


LL2-02 subtractionSC vs. AA4' BTK associated


beige-related DDt dill. AAd' vs. AA4' contains WD repeat
proteins 16 display


Transcriptional SA.i9PlsubtractionSC vs. AAd' polyeomb homol.
regulators


LLS-96 subtractionSC vs. AA4' includes LIM domain


G-protein signalingLL.t-39subtractionSC vs. AA4' cell cycle transition


B l-66 subtractionSC vs. AA.t' similu to BL34


Apoptosis-relatedSBSA56 subtractionSC vs. AA4' SVtT3A-related
genes


LL3-68 subtractionSC vs. AA4' sim. to Requiem
transcript. factor


Chromatin proteinsC.a-23 subtractionSC vs. AA4' yeast HST3like


C3-25 subtractionSC vs. AA4' NHP2like


LL2-89 subtractionSC vs. AAd' yeast SIS2-like


Other stem cells B3-T7 subtractionSC vs. AAi' homologous to A4
(intestinal gene


crypts) C2-48 subtractionSC vs. AA.t- homologous to C101


LL2-76 subtractionSC vs. AAd' homologous to EDPF


Genes involved LLS-03 subtractionSC vs. AAd' t(1:11)(q21:q23)
in leukemogettic


translocations B2-67 subtractionSC vs. AA4' t(X:14)(q28:q11)


Homologues of LL235 subtractionSC vs. AAt' germ cell-less
Drosophila


developmental LL2-l2 subtraction5C vs. AA4' brainiac
gents


C.t-$0 subtractionSC vs. AA.t' cornichon


B.i-1s subtractionSC vs. AA4' discs-large


~The dcsignanon "SC" refers to sorted day Lt fetal liver cells having the
phenotype AAi. I' Lin''Sca cktt'.


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FI~A_MPL li; 2: Molecular cloning and characterization of AA4, an early
marker of hematopoietic development
In this example, the expression cloning and molecular characterization of AA4,
a
surface marker expressed on hematopoietic stem and progenitor cells is
described. The
results demonstrate that AA4 is a 130kDa type I glycosylated membrane protein
whose
structural organization suggests a role in cell adhesion. Expression analysis
showed that
high levels of AA4 are found in lung, heart, and bone marrow. It is not found
in
undifferentiated ES cells, but its expression is upregulated as these cells
differentiate into
colonies of hematopoietic precursors and endothelial cells. In the
hematopoietic system,
t0 expression of AA4 correlates with the expression of stem cell markers CD34
and CD43.
Functional studies revealed that AA4 coimmunoprecipitates with CD34 and CD43,
suggesting that these proteins form a macromolecular complex which functions
in the
regulation of cell adhesion, proliferation and/or differentiation of
hematopoietic cells.
Introduction
Monoclonal antibody AA4.1 was first described more than a decade ago
(McKearn et al., 1984) and since then it has become a useful marker for the
isolation and
analysis of hematopoietic cells (McKearn et al., 1985; Jordan et al., 1990;
Fujimoto et al.,
1996). A number of works have shown that the antigen recognized by AA4.1 is
present
on a subset of primitive hematopoietic progenitors found at various stages of
development
2o in sites of active hematopoiesis in yolk sac (Cumano et al., 1993; Auerbach
et al., 1996;
Yoder et al., 1997), AGM region (Godin et al., 1995; Marcos et al., 1997),
fetal liver
(McKearn et al., 1985; Jordan et al., 1990; Cumano and Paige, 1993), and bone
marrow
(Li et al., 1996; Szivassy and Cory, 1993). In yolk sac, AA4-positive cells
are first
detected at day 8-10 of gestation (Cumano et al., 1993; Sanchez et al., 1996).
At day 9-10
of gestation AA4+c-Kit+ progenitors are found in the P-Sp/AGM region (Sanchez
et al.,
1996; Marcos et al., 1997), and by day 14 of development, AA4 defines 0.5-1.0%
of the
fetal liver tissue that contains the entire hierarchy of primitive
hematopoietic cells (Jordan
et al., 1990). Proliferation within each successive compartment results in
increased total
number of progenitor cells. Antigen density per cell also increases with
developmental
3o progress, which is especially marked for c-Kit and AA4 (Marcos et al.,
1997). In bone
marrow, HSC are found in both AA4+ and AA4- subpopulations, although in adult
marrow AA4 is largely regarded to be a marker of early B lymphoid lineage. In
addition,


CA 02340465 2001-02-21
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recent studies demonstrate that the expression of AA4 parallels the onset of
hematopoietic
development in differentiating ES cells (Kabrun et al., 1997; Lin and Neben,
1997;
Potocnik et al., 1997). Taken together, these results indicate that AA4 plays
an important
role in hematopoiesis and has to be studied in more detail.
In order to achieve this goal, AA4 was molecularly cloned and characterized.
The
results demonstrate that AA4 is a 130kDa type I transmembrane glycoprotein
whose
structure suggests a role in cell adhesion. Expression analysis showed that
high levels of
AA4 are found in lung, heart, and bone marrow. In the hematopoietic system,
expression
of AA4 correlates with the expression of stem cell markers CD34 and CD43.
Functional
1o studies indicate that AA4 coimmunoprecipitates with CD34 and CD43,
suggesting that
these proteins form a macromolecular complex which may function in the
regulation of
cell adhesion, proliferation and/or differentiation of hematopoietic cells.
Results
Cloning n, f AA4. In order to identify AA4, AA4.1 monoclonal antibody was used
to screen various cell lines and primary hematopoetic cells. The murine B
lymphoid cell
line D2N was found to expresses relatively high levels of AA4 antigen (see
Table 3).
Immunoprecipitation of protein extracts prepared from D2N cells showed that
AA4.1
recognizes a protein with apparent molecular weight (Mr) 130kDa (Figure 7).
This
protein was also present in extracts prepared from B lymphoid cell line M2.4,
and AA4
2o positive hematopoietic cells derived from bone marrow and fetal liver.
To isolate AA4, a cDNA library was prepared from D2N cells and cloned into the
polylinker region of a retroviral expression vector REBNA (see Materials and
Methods).
Following production of retroviruses, NIH 3T3 cells were infected with the
recombinant
cDNA library and selected for AA4 expression by flow cytometry using PE-
conjugated
2s AA4.1. After two rounds of sorting, genomic DNA extracted from AA4-positive
cells
was analyzed by pcr amplification using viral vector primers. This resulted in
the
amplification of a 3.1 kbp cDNA which was gel-purified and subcloned for
further
analysis. Infection of NIH 3T3 fibroblast or EML C1 hematopoietic cells with
REBNA/AA4, a recombinant retrovirus expressing the cloned cDNA, has lead to
the
3o acquisition by cells of high affinity to AA4.1 mAb (Figure SA). In infected
cells, AA4.1
detects a 130kDa surface protein which comigrates with the endogenous AA4 from
D2N
cells (Figure 8B), thus indicating that the cloned cDNA encodes AA4.


CA 02340465 2001-02-21
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Seauence analvsis ofAA4. Sequence analysis of the cloned cDNA (SEQ. ID
No.: 72) showed that it has a single open reading frame encoding a protein of
644 amino
acids (see Figure 9)(SEQ. ID No.: 73. The deduced amino acid sequence includes
a
putative leader peptide and the mature protein which starts at position 20.
The protein
contains a long N-terminal extracellular region, a single putative hydrophobic
transmembrane region, and 47 amino acids of the C-terminal cytoplasmic domain.
The
extracellular part of AA4 is composed of two major structural moieties. The N-
terminal
region contains a C-type lectin domain (CTL) which has 32% sequence homology
to
endothelial cell receptor thrombomodulin. This region is followed by a
cysteine-rich
t0 domain composed of six epidermal growth factor (EGF)-like repeats, three of
which are
consistent with the calcium-binding EGF motifs. Similar repeats are found in
the
extracellular domains of a large number of membrane-bound proteins and in
proteins
known to be recreated (Bork et al., 1996).
Database searches revealed that AA4 exhibits high homology to C 1 qRP, the
is human receptor for complement component Clq expressed on surfaces of
myeloid cell
lineage and endothelial cells (Nepomuceno et al., 1997). Sequence alignment
showed that
AA4 and C 1 qR have approximately 68% identical amino acid positions and
similar
domain structures. Highest homologies were found within the N-terminal parts
of the
proteins and their C-terminal cytoplasmic domains (see Figure l0A and lOB),
suggesting
20 that AA4 and C 1 qR may share functional properties.
The amino acid sequence of AA4 contains numerous potential O-linked and N-
glycosylation sites. Although the predicted Mr of AA4 is 67.4 kDa, two protein
bands
exhibiting Mr lOSkDa and 130kDa respectively, are immunoprecipitated by AA4.1
mAb
in cells infected with REBNA/AA4 (Figure 8C). When 35S-labeled cells are
chased with
25 nonradioactive media, the intensity of the lOSkDa band rapidly decreases,
while the
intensity of the 130kDa band increases, thus indicating that p130 is the
mature form of the
protein, whereas p 1 OS is its precursor. In agreement with this conclusion,
immunoprecipitation of biotinyated cells using AA4.1 reproducibly results in
the
detection of a 130kDa surface protein (see Figure 8B).
30 ~xinression inatterns ofAA4. Northern blot analysis showed that in adult
mouse
tissues, AA4 is expressed at high levels in lung, heart, and bone marrow. No
detectable
expression was found in brain, testis, spleen, and thymus (Figure 10B). In
normal tissues


CA 02340465 2001-02-21
WO 00/11168 47 PCT/US99/19052
and transformed cell lines, a 7kb RNA species hybridizes with the cloned cDNA
(Figure
11A and 11B). In addition to the 7kb species, poly(A)-RNA from D2N cells
contains a
minor band corresponding to a 3.2 kb mRNA (see Figure 11A, lane 8). Similarity
search
against expressed sequence tags (ESTs) showed that databases contain at least
seven
different sequences corresponding to the 3'-untranslated region of the cloned
aa4cDNA
which were isolated from the following mouse tissues: colon (Genbank accession
No
AA929174), heart (AA435107), lymph node (AA185911 and AA267407), lung
(AA220480), mammary gland (AI021507), and spleen (AA145088). Sequence identity
with the ESTs abrogates upstream of nucleotide G at position 2481 in the 3'-
untranslated
1o region of aa4cDNA, thus indicating that the cloned cDNA corresponds to an
alternatively
spliced 3.2 kb aa4mRNA.
~;xnression ofAA4 in hematonoietic ~ro~enitor cells AA4 is produced in murine
hematopoietic progenitor cells and immature B lymphocytes found at various
stages of
development in yolk sac (Godin et al., 1995; Marcos et al., 1997), fetal liver
(MeKearn et
t 5 al., I 985; Jordan et al., 1990), and bone marrow (Cumano et al., 1992; Li
et al., 1996). To
confirm that the cloned cDNA encodes AA4, RT-PCR was performed on
hematopoietic
cells fractionationated using several different techniques. The cells analyzed
included
AA4+ and AA4- fetal liver (FL) cells; AA4+ FL cells fractionated into Linloc-
Kit+Sca-
1+ and Linloc-Kit+Sca-1- populations; and hematopoietic progenitors isolated
from bone
2o marrow (BM) by cell sorting using combinations of different surface
markers.
Figure 12A shows that aa4 was amplified from AA4+ FL cells, whereas in AA4-
cells it was only present at low levels. AA4 expression was high in Linloc-
Kit+Sca-1+
cells enriched for HSC activity. In adult marrow cells, aa4 was amplified from
Lin+ cells
and Lin-c-Kit+Sca-1+CD34+ multipotential progenitors. At lower levels aa4 was
present
25 in Lin-c-Kit+Sca-1+CD34- long term reconstituting HSCs (Figure 12B). In all
tested cells
aa4 expression correlated with the expression of stem cell markers CD34 and
CD43.
Embryonic stem (ES) cells which have been shown to generate progenitors for
most hematopoietic lineages during differentiation in vitro (Keller, 1995)
were also tested
in this experiment. Figure 12C shows that aa4 was not found in
undifferentiated ES cells
3o but its expression was upregulated as these cells differentiated and formed
blast cell
colonies (BL) and colonies of more differentiated hematopoietic cells (HMT).
These
results are in line with previous studies which showed that AA4 is expressed
in ES-


CA 02340465 2001-02-21
WO 00/11168 48 PCT/US99/19052
derived hematopoietic precursors (Kabrun et al., 1997; Ling and Neben, 1997;
Scott et al.,
1997) and show that this expression is not abrogated upon differentiation of
blast cell
colonies into endothelial cells (ENT in Figure 12C).
Fcto~ic xnressiorr ~,f~~has no mitoQerric effect EML C1 and NIH 3T3 cells
expressing exogenous AA4 did not exhibit morphological changes indicative of
alterations in their growth properties. To examine the effect of AA4 on growth
phenotypes in more detail, NIH 3T3 and primary mouse embryo fibroblasts were
infected
with a retrovirus expressing AA4 and maintained in high and low serum
conditions.
FACS analysis of the transduced cells confirmed that in each case the
efficiency of
to infection was close to 100%. However, examination of growth rates showed
that
overexpressed AA4 had no apparent effect on proliferation of both cell types
as compared
to control uninfected cells or the corresponding cells infected with a
retrovirus expressing
GFP.
~A4 coimmrmonrecinitates mith CD34 and CD43 To investigate interactions
~ 5 with other proteins, hematopoietic and fibroblast cells expressing AA4
were
imrnunoprecipitated with AA4.1 mAb and then examined by immunoblot analysis
using a
panel of antibodies directed against membrane-associated proteins. This
analysis revealed
that AA4 coimmunoprecipitates with CD34, a membrane glycoprotein selectively
expressed within the hematopoietic system on stem and progenitor cells, and
CD43 which
2o is a major O-glycosylated sialomucin found on the surfaces of most
leukocytes. This
result is in line with previous studies which showed that CD43
coimmunoprecipitates
with the human C 1 qRP (Guan et al., I 991; 1994). Figure 13B shows that in
the murine
D2N and EML C1 cells, a 52kDa protein is the major isoform of CD43 that
associates
with AA4. This 52kDa protein was found to be reactive with both the N-terminal
(S 19)
25 and C-terminal (M19) anti-CD43 antibodies, indicating that it is not a
breakdown product.
In NIH 3T3 fibroblasts coexpressing CD43 and AA4, a 54kDa and a 170kDa CD43
isoforms coimmunoprecipitated with AA4 pointing to the glycosylation
differences
brtween CD43 expressed in different cell types. A 115kDa CD43 isoform which
previously have been shown to be sialylated and thus overly negatively charged
(Guan et
3o al., 1994) did not form macromolecular complexes with AA4 in NIH 3T3 cells.
Similarly,
AA4 did not coimmunoprecipitate with a 115kDa CD43 isoform found in D2N cells
and
a 120kDa isoform found in EML C1 (Figure 13B).


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To examine associations of AA4 with CD34, both proteins were expressed in Rat-
1 cells following infection with the corresponding retroviruses. See Figure
13C.
In Rat-1 cells coexpressing AA4 and CD34, AA4.1 the results also demonstrate
that AA4 coimmunoprecipitates with CD34. Studies have suggested a role for
both CD34
and CD43 in the regulation of adhesion, growth and differentiation of
hematopoietic
precursors (Ardman et al., 1992; Bazil et al., 1997; Chen et al., 1996; Suzuki
et al., 1996;
Wood et al., 1997; Stockton et al., 1998). Coexpression of AA-Fc fusion
protein in NIH
3T3 cells stably producing AA4 showed that AA4-Fc forms heterodimeric
complexes
with AA4. Figure 11A shows that equimolar amounts of AA4 and AA4-Fc were
to coprecipitated from these cells by protein A, indicating that AA4 is prone
to homo- or
heterodimerization.
Discussion
This Example describes the expression cloning and sequence analysis of AA4, a
molecular marker expressed on hematopoietic stem and progenitor cells. The
CDNA
encoding AA4 was isolated from a retroviral cDNA library prepared from the
murine
D2N lymphoid cell line. Sequence analysis of the cloned eDNA revealed that AA4
is a
type I transmembrane protein composed of 625 amino acids. The extracellular
part of the
molecule contains two major structural moieties, a C-type lectin carbohydrate
recognition
domain and six EGF-like domains. Similar repeats have been found in a large
number of
2o membrane-bound proteins or in proteins known to be secreted. The
cytoplasmic domain
of AA4, in contrast, bears no structural similarity with known protein
families. Instead,
AA4 revealed strong homology to CIqR, the human receptor for complement
component
Clq which is predominantly expressed in phagocytic cells such as monocytes,
neutrophils,
and endothelial cells (Nepomuceno et al., 1997). Sequence alighnment shows
that AA4
and CIqR have approximately 68% identical amino acid positions and share
similar
domain structure. Highest homologies were found within the Nterminal parts of
the two
proteins and their C-terminal cytoplasmic domains, suggesting that AA4 and
CIqR may
have similar functional properties. Expression of AA4 correlates with the
expression of
CD34 and CD43, two other surface markers normally present on murine
hematopoietic
3o stem and progenitor cells. Previous studies have shown that AA4-positive
cells are first
detected in yolk sac at day 8-10 of gestation (Cumano et al., 1993; Sanchez et
al., 1996).
At day 9-10 pc AA4+c-Kit+Mac-I+ progenitors are found in the P-Sp/AGM
region (Sanchez et al., 1996; Marcos et al., 1997), and by day 14 of
gestation, AA4


CA 02340465 2001-02-21
WO 00/11168 50 PCT/US99/19052
defines 0.5-1.0% of the fetal liver tissue that contains the entire hierarchy
of primitive
hematopoietic cells (Jordan et al., 1990). As proliferation within each
successive
compartment results in increased total number progenitor cells, antigen
density per cell
also increases with developmental progress, which is especially marked for c-
Kit and
s AA4 (Marcos et al., 1997). A significant proportion of hematopoietic
progenitors from
yolk sac also express CD34, CD38, CD43, CD44, and Mac-1 however show little or
no
expression of CD4, CDB, CD45R and Sca-. These same markers are present on
FLderived
HSC which also express Sca-1. Adult marrow HSCs also express cKit, CD38 and
Sca-1,
but do not normally express Mac-I or AA4. In BM, HSC are found in both AA4+
and
to AA4-subpopulations, although in adult marrow AA4 is largely regarded to be
a marker of
early B lymphoid lineage 0. These results indicate that the expression of cell
surface
antigens changes on HSC during ontogeny and that differential display of
theses cell
surface markers may reflect relationship between HSCs that contribute to
multilineage
hematopoiesis and distinct anatomical sites during development.
15 Methods
~.11s and tissue culture. NIH 3T3 fibroblasts were grown in DME medium
supplemented with 10% fetal calf serum (FCS). D2N cells were grown in RPMI
1640
medium containing 10% FCS. EML C1 cells were grown in IMDM supplemented with
20% horse serum and 8% BHK/MKL conditioned medium (Tsai et al., 1994). To
2o maintain the multipotentiality of EML Cl, the cells were kept at low
density (0.5 - 5x10-
5/ml) and subcultured every two days. Cell lines constitutively expressing AA4
and GFP
were derived from NIH 3T3 or EML Cl cells by infecting with the corresponding
recombinant retroviruses.
Retroviral-mediated gene ~ n_~. Retrovirus expression vector REBNA was
25 constructed by substituting the LacZ gene contained within the EcoRI-Notl
fragment of
plasmid LZRSPBMN-Z (Kinsella and Nolan, 1996) with a synthetic polylinker
composed
of EcoRl, Xhol, Sfil, and Notl sites. Retroviral vector REBNA/IRESGFP contains
a
poliovirus IRES element and the CDNA encoding color-enhanced GFP (S.
Zolotukhin,
Gainesville, FL) inserted into REBNA.
30 For DNA transfections, cells were plated at a density 2x106 cells per 60mm
dish
and transfected with Sug of plasmid DNA using 20u1 of lipofectamine reagent
(GibcoBRL). REBNA-transfected cells were selected in puromycin (2ug/ml) and
grown
to confluence prior to collecting virus supernatant. For infections" the
culture medium


CA 02340465 2001-02-21
WO 00/11168 51 PCTNS99/19052
was supplemented with polybrene (Sigma) at Sug/ml. The appropriate virus was
added
and incubated overnight. Multiple infections were carried sequentially, with
the
appropriate selection between.
Isolation o.t RNA and CDNA cloning. Poly(A) RNA prepared from D2N cells
s was converted into CDNA using Superscript II Reverse Transcriptase
(GibcoBRL) and an
oligo(dt) primer containing NotI site, S'-TGGTGTCGACGCAGAGTAGCGGCCGCT18
(SEQ.ID.No.:74). The second strand was synthesized using DNA polymerase I in
combination with E. coli RNAse H and E. coli DNA lipase as described (Gubler
and
Hoffman, 1983). An adaptor composed of complementary oligonucleotides, S'-
to GGCCCGGGCCGGCC (SEQ.ID.No.:75) and S'-TCGAGGCCGCCCGGGCC
(SEQ.ID.No.:76), was ligated to the CDNA and cut with Notl to produce CDNA
molecules with NotI and Xhol termini for directional cloning. After size
fractionation in
agarose gel, eDNAs larger than 2.5 kbp were Iigated into Notl and XhoI cut
plasmid
REBNA and electroporated into electrocompetent DH12S cells (GibcoBRL). Plasmid
15 DNAs were transfected into 293-derived packaging cell line for retrovirus
production.
Virus-containing supernatants were collected and stored at -80C.
NIH 3T3 cells infected with the recombinant retroviruses representative of D2N
CDNA library were selected for the production of AA4 by flow cytometry using
phycoerythrin-conjugated AA4.1 mAb. After two rounds of sorting, genomic DNA
2o isolated from AA4-positive cells was subjected to pcr amplification using
retroviral
vector primers, S'CAGCCCTCACTCCTTCTC (SEQ.ID.No.: 77) and S'-
GGTGGGGTCTTTCATTCC (SEQ.ID.No.: 78) (Kitamura et al., 1995). Amplified
CDNA was gel purified and subcloned into pbluescript SK and REBNA plasmid
vectors.
Nucleotide sequences were analyzed using NCBI Blast database search programs
and
25 ExPASy molecular biology server from the Swiss Institute of Bioinformatics.
Northern blot, hybridization. RNAs prepared using acid guanidinium thiocyanate-

phenol extraction procedure (Chomczynski and Sacchi, 1987) were separated.on
formaldehydeagarose gels and blotted onto the Hybond-N nylon membranes
(Amersham). Hybridization probes were derived from cloned cDNAs using Ready To
3o Go DNA labeling beads {Pharmacia Biotech). Hybridizations were performed as
described previously (Petrenko et al., 1997).
Cell labeling and Immunonrec_ipitations. The ECL protein biotinylation system
(Amersham) for the detection of cell surface proteins was used as recommended
by the


CA 02340465 2001-02-21
WO 00/11168 52 PCT/US99/19052
manufacturer. For radioactive labeling, 2x106 cells were incubated with 200uCi
Translabel (ICN) in 2m1 of cysteine and methionine-deficcient medium for 2hr
at 370C.
Cells were washed in PBS and lyzed l Omin. on ice in 500u1 of NP40 buffer
containing
2OmM TrisHCI pH 7.6; ISOmM NaCI; 0.5% NP40; ImM PMSF; SmM benzamidine;
ImM sodium vanadate; LOug/ml aprotinin; IOug/ml leupeptin. Lysates were
cleared by
centrifugation and tug of soluble antibody were added to the supernatants
followed by
25u1 of Protein G-Sepharose suspension (Sigma). After 2 to 4hr incubation at
40C with
rotation, protein Gantibody complexes were pelleted and washed in the
successive
changes of wash buffer I (1M NaCI; IOmM TrisHCI pH 8; 0.1 % NP40); wash buffer
2
(O.IM NaCI; IOmM TrisHCI pH 8; 0.1 % NP40); wash buffer 3 (IOmM TrisHCI pH 8;
0.1 % NP40). Samples were eluted by boiling 2min. in 2Xge1 loading buffer,
separated by
SDS/PAAG, dried and exposed to X-ray film.
Western blot analysis. Protein extracts for Western blot analysis were
prepared as
described (Mornson et al., 1991 ). The antibodies used included goat anti-
mouse CD43
~5 polyclonal IgG (M19 and S-19, Santa Cruz) in combination with HRPconjugated
secondary antibodies and ECL detection system (Amersham).
Flow c_~~~e~ and RT-PCR. Timed-pregnant mice and 5- to 7-week-old female
mice (C57BI/6j) were purchased from Jackson Laboratory (Bar Harbor, ME). AA4-
positive cells were isolated from day 14 fetal liver by immunopanning on Petri
dishes
2o coated with AA4.1 antibody (l0ug/ml). Hematopoietic stem cells were
purified from
AA4-positive fraction by staining with lineage-specific antibodies as
described previously
(Moore et al., 1997). Three-color fluorescence-activated cell sorting for
lineage negative
to low, Sca-I(+), c-Kit(+) cells was performed on a multilaser FACS Vantage
with
CeIIQuest software (Beckton Dickinson). ES cells differentiated into blasts
cell colonies,
25 hematopoietic progenitors, and endothelial cells prepared as described
previously.
For RT-pcr, poly(A)-RNA isolated from sorted cells was converted into CDNA
using Superscript II Reverse Transcriptase (GibcoBRL) and CapFinder CDNA
amplification kit (Clontech). Gene-specific primers for pcr amplification
included: 5'-
TTCAGCAAGCCCTGACTC (SEQ.ID.No.:79) and 5'GCCACCTTCGAAGCAATC
30 (SEQ.ID.No.:80) (AA4); 5'-GAGCGGTACAGGAGAATG (SEQ.ID.No.:81) and
5'GCCCACCCAACCAAATCA (SEQ.ID.No.:82) (CD34); 5'-
ACCGCGTTCTTCTGTAAC (SEQ.ID.No.:83) and S'CAGCTAACAGCAGGATCC


CA 02340465 2001-02-21
WO 00!11168 53 PCT/US99/19052
(SEQ.ID.No.:84) (CD43); G3PDH Control Amplimer Set (Clontech) for the
amplification
of GAPDH.


CA 02340465 2001-02-21
WO 00/11168 54 PCTNS99/19052
EXAMPLE 3: IN VITRD MAINTENANCE OF HIGHLY PURIFIED,
TRANSPLANTABLE HEMATOPOIETIC STEM CELLS
The cellular and molecular mechanisms which regulate even the most primitive
hematopoietic stem cell are not well understood. This example details a
systematic
dissection of the complex hematopoietic microenvironment in order to define
some of these
mechanisms. An extensive panel of immortalized stromal cell lines from murine
fetal liver
was established and characterized. Collectively, these cell lines display
extensive
heterogeneity in their in vitro hematopoietic supportive capacities. This
example describes a
t0 long-term in vitro culture system, utilizing a single, stromal cell clone
(AFT024) that
qualitatively and quantitatively supports transplantable stem cell activity
present in highly
purified populations. Disclosed is multi-lineage reconstitution in mice that
received the
equivalent of as few as 100 purified bone marrow and fetal liver stem cells
which were
cultured for 4-7 weeks on AFT024. The cultured stem cells meet all functional
criteria
currently ascribed to the most primitive stem cell population. The levels of
stem cell activity
present after 5 weeks of coculture with AFT024 far exceed those present in
short-term
cytokine-supported cultures. In addition, the maintenance of input levels of
transplantable
stem cell activity is accompanied by the expansion of other classes of
stem/progenitor cells.
This suggests that the stem/progenitor cell population is actively
proliferating in culture and
2o that the AFT024 cell line provides a milieu which stimulates progenitor
cell proliferation
while maintaining in vivo repopulating activity.
Introduction
Mammalian blood formation originates in a small population of hematopoietic
stem
cells. The hallmark features of these cells are: (1) a hierarchical
multilineage differentiation
potential with the ability to clonally give rise to at least 8 distinct cell
lineages, (2) self
renewal capacity which is reflected in the life long continuous activity of
few, in some cases
single, stem cells and (3) a dramatic proliferative potential which is
ultimately responsible
for the production of large numbers of mature blood cells. (Leminschka, LR.
1992;
Morrison, S.J. et al. 1995; Harrison, D.E. 1980). During the past decade much
progress has
3o been made in providing a physical phenotype for this rare population of
stem
cells.(Spangrude, G.J., et al. 1995; Lemischka, LR. 1992; Bauman, J.G., et al.
1998).
However, currently, the only reliable functional assay system for the most
primitive stem
cell compartment is long-term in vivo transplantation. No in vitro system has
been


CA 02340465 2001-02-21
WO 00/11168 55 PCT/US99/19052
developed which adequately recapitulates stem cell behaviors. Therefore, the
cellular and
molecular mechanisms that regulate the biology of stem cells have remained
obscure.
A major challenge in stem cell research is the establishment of culture
systems which
facilitate in vitro maintenance of long-term transplantable stem cell
activity. This is a
necessary first step towards a cellular and molecular understanding of the
regulatory
mechanisms which mediate commitment versus self renewal decisions. Moreover,
the
establishment of such culture systems is a prerequisite for the potential
expansion of
undifferentiated stem cell populations as well as for the generation of
stemlprogenitor cells
committed to selected lineages.
to Efforts to develop culture systems for the maintenance of transplantable
stem cells
can be subdivided into two broad categories: ( 1 ) those utilizing defined
cytokine
combinations as the only culture supplements and (2) those relying on a pre-
established
stromal monolayer as an additional supportive component (with or without
exogenously
added cytokines). Both of these strategies have met with only limited success.
In the first
case, it has been repeatedly demonstrated that combinations of cytokines can
exert potent
stimulatory effects on stem/progenitor populations. (Spangrude, G.J., et al.
1988; Jordan,
C.R. et a(. 1990; Fleming, W.H., et al. 1993). In some studies, highly
purified stem cells (Li,
C.L., and Johnson, G.R. 1990; Spangrude, G.J. and Johnson, G.R. 1990). have
been used
and the direct effects of cytokines have been demonstrated at the single cell
level. (Jones, R.
2o et al. 1990; Uchida, N. et al. 1993). While informative, the vast majority
of these studies are
limited by their strictly in vitro nature. Thus, it is feasible to expand,
replatable in vitro
progenitor populations (Li, C.L., and Johnson G.R. 1992; Uchida, N. et al.
1993) and to
stimulate colony-formation by cells with both myeloid-erythroid and lymphoid
potentials,
Jones, R., et al. 1990; Uchida, N. et al. 1993; Trevisan, M., and Iscove, N.N.
1995; Ogawa,
M. 1993) however, the equivalence of these progenitor cells with the in vivo
transplantable
stem cell population remains speculative. Several studies have clearly
demonstrated a
dramatic loss of in vivo repopulating potential as a result of cytokine driven
in vitro
proliferation.(Knobel, et al., 1994; Peters, et al., 1995; Traycoff, et al.,
1996). A small
number of studies have shown that defined cytokine combinations promote the
maintenance
of transplantable activity.(Rebel., et al., 1994). However, most of these are
limited both by
the use of very short culture periods, the exact nature of the in vivo assay,
and the use of
non-enriched stem cell sources.(Muench, et al., 1993; Holyoake, et al., 1996;
Soma, et al.,


CA 02340465 2001-02-21
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1996). This precludes interpretations suggesting a direct action of the given
cytokine(s) in
maintaining transplantable activity.
A further complication with defined cytokine studies is the inability to
ascribe in vivo
physiological relevance to the observed effects. It has long been accepted
that in the intact
animal, stem cells are found in close association with discrete cellular
microenvironments.(Lord, et al., 1975; Trentin, et al., 1970; Weiss, et al.,
1991; Wolf, 1979).
These observations suggest both the existence of stem cell niches and the
notion that in vivo
stem cell regulatory mechanisms are likely to require cell-cell contact or
short range
interactions.(Dorschking, 1990). Efforts to understand the features of the
hematopoietic
to microenvironment began with the establishment of the Dexter long-term
culture (LTC)
system. (Dexter, et al., 1977). In this culture system hematopoiesis is
maintained for weeks
or months by a heterogeneous adherent cell monolayer derived from bone marrow
(BM).
While some degree of transplantable stem cell maintenance and self renewal
(Eraser, et al.,
1990) has been demonstrated, a general feature of the Dexter-LTC is a dramatic
net decrease
t5 of stem cell activity over time. (Harrison, et al., 1987; Van der Sluijs,
et al., 1993). Although
much progress has been made, especially in studies of human stem/progenitor
cells,
(Sutherland, et al., 1989; Verfaillie, et al., 1995; Hao, et al., 1996) a
further drawback of this
system is the heterogeneity of the stromal cell types present in the
supportive monolayer.
This hampers the identification of regulatory mechanisms. Studies have been
reported
zo where the heterogeneous stromal monolayer is replaced with cloned stromal
cell lines.
(Roberts, et al., 1987; Kodama, et al., 1984; Issad, et al., 1993; Wineman, et
al., 1993). Many
of these cell lines can support in vitro myelopoiesis, (Suzuki, et al., 1992;
Neben, et al.,
1993; Kodama, et al., 1992) B-lymphopoiesis (Collins, et al., 1987; Whitlock,
et al., 1982)
or in some cases both.Wineman, et al., 1993; Wineman, et al., 1996) However,
very few
25 studies have focused on the in vitro maintenance of the most primitive
transplantable stem
cell compartment. Moreover, with one exception (Szilvassy, et al., 1996) the
studies which
have focused on the in vitro maintenance of this stem cell population begin
with
heterogeneous unpurified sources of hematopoietic activity. Wineman, et al.,
1992, 1996;
Deryugina, et al., 1994). Such populations contain numerous non-hematopoietic
stromal cell
3o types. Therefore, it has not yet been possible to assign a direct stem cell
supporting
phenotype to a given stromal cell line.
It was hypothesized that the rare frequency of primitive, stem cells may
suggest an
equally rare frequency of stem cell supporting microenvironmental niches.
Accordingly, we


CA 02340465 2001-02-21
WO 00/11168 S7 PCT/US99/19052
established and characterized a large panel of conditionally immortalized,
cloned stromal
cell lines from mid-gestation fetal liver. This organ was chosen because,
during
development, it is here that the stem cell compartment is undergoing self
renewal expansion
in addition to differentiation. (Moore, et al., 1970). The cell lines were
generated as
previously described, (Wineman, et al., 1996) by immortalization with a
temperature
sensitive SV40TAg. (Frederiksen, et al., 1988) The clonal nature of the
AFT024, 2018, and
2012 cell lines was verified by Southern blot analysis which detected a
single, unique
proviral integration locus in their genomic DNA. In order to identify
potentially interesting
cell lines, we used a "cobblestone area" (CA) assay (Ploemacher, et al., 1989)
was initiated
1o with BM taken from mice injected two days previously with 5-fluorouracil (5-
FU). It has
been suggested that CA colonies which appear after a prolonged culture period
are derived
from more primitive stem cells, possibly identical to some in vivo
transplantable entities.
(Ploemacher, et al., 1991 ). Therefore, a goal was in identifying cell lines
which support such
late arising CAs. Of 225 lines, 77 (34%) were capable of supporting limited in
vitro
hematopoiesis, while, consistent with the initial hypothesis, only 2% were
able to maintain
long-term (>6 weeks) hematopoietic CA activity. Subsequent studies with a
selected subset
of these lines, showed that the ability to effectively support in vivo
reconstituting BM stem
cells is infrequently observed. (Wineman, et al., 1996). Two out of sixteen
cell lines
maintained significant levels of long-term reconstituting stem cell activity
for an in vitro
2o culture period of three weeks. Several other cell lines supported low
levels of such activity
or transiently repopulating stem cells. The cell inoculum, in these studies,
was whole BM
which was not enriched for stem cell activity. Therefore, it was not possible
to suggest that
the effective stromal cell lines were directly supporting stem cell activity.
This example
demonstrates that a single clonal cell line, designated AFT024, can maintain
quantitative
levels of transplantable stem cell activity present in highly purified stem
cell populations.
These data were generated using a competitive repopulation assay system, which
employs
uncornpromised competitor BM cells. The in vitro-maintained stem cells satisfy
all criteria
which currently define the most primitive stem cell population including the
ability to
reconstitute secondary recipients. This example also shows that the in vitro
maintenance of
3o primitive transplantable stem cells is compatible with the concurrent
generation of large
numbers of committed progenitors.


CA 02340465 2001-02-21
WO 00/11168 58 PCTNS99/19052
Methods
Mice. Timed-pregnant mice and 5-7 weeks old females (C57BI/6J, Ly5.2) were
purchased from the Jackson Laboratory (Bar Harbor, ME). Congenic C57B1/6,
Ly5.1
female mice were purchased from the National Cancer Institute (Frederick, MD).
All mice
were housed in the Princeton University Barrier Animal Facility, in autoclaved
micro-
isolator cages on ventilated cage racks. The animals received sterile,
irradiated food, and
acidified, autoclaved water ad libitum.
Stromal cell lines and culture conditions. The fetal liver stromal cell lines
used in
this study were derived as previously described. (Wineman, et al., 1996).
Stromal cell lines
to were routinely cultured in Dulbecco's modified Eagles's medium (DMEM)
supplemented
with 10% fetal bovine serum (FBS), SX10-5 mol/L b-mercaptoethanol (2-ME), at
32°C, 5%
CO2, 100% humidity. Sera were obtained from Hyclone, Logan, UT. Other
biochemical
reagents were obtained from Sigma, St. Louis, MO. Two of the lines used in
this study
(2012 and 2018) were previously characterized for their ability to support
long-term
repopulating activity present in whole unfractionated BM. (Wineman, et al.,
1996). The
AFT024 cell line was identified as an additional long-term (>4 weeks) CA
supporter.
Subclones of 2012 and AFT024 were isolated and used in these studies. The
AFT024 cell
line has remained stable and demonstrated consistent stem cell supporting
abilities for over 4
years.
2o Hematopoietic sterrL~~~urification. Stem cells were purified from day 14
fetal
livers essentially as described, (Jordan, et al., 1990) with the inclusion of
c-kit expression
as an additional parameter. Briefly, AA4.1+ cells were isolated by
immunopanning on
petri dishes coated with AA4.1 antibody (10 ug/mL). The AA4.1+ fraction has
been
shown to contain all repopulating stem cell activity present in day 14 fetal
liver. (Jordan,
et al., 1990) AA4.1+ cells were collected and stained with saturating
concentrations of
fluorescein isothiocyanate (FITC) labeled rat monoclonal antibodies to lineage
markers
(CD3, CD4, CDS, CDB, B220, Gr-1, Mac-1, and TER-119). The cells were
simultaneously stained with phycoerythrin (PE) labeled Ly6A/E (Sca-1) antibody
and
biotinylated antibody to c-kit. The latter was developed with streptavidin
3o allophycocyanin (APC). The AA4.1 hybridoma was a kind gift from Dr. J.
McKearn,
Monsanto, St. Louis, MO. AA4.1 antibody was purified by ImClone Systems Inc.
New
York, NY. The TER-119 antibody was initially obtained from Dr. T. Kina, Kyoto
University, Japan and subsequently purchased from PharMingen, San Diego, CA.
All


CA 02340465 2001-02-21
WO 00/11168 59 PCTNS99/19052
other antibodies were purchased from PharMingen. Streptavidin APC was
purchased
from Molecular Probes Inc., Eugene, OR. Three color fluorescence activated
cell sorting
for lineage negative to low (lin'"°), Sca-1+, c-kit+ cells was
initially done on a dual laser
Epics 753 cell sorter (Coulter Electronics, Hialeah, FA) interfaced with
Cicero software
(Cytomation Inc., Fort Collins, CO) and subsequently on a mufti-laser FACS
Vantage
with Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose,
CA).
Enriched fractions of BM stem cells were obtained from CS7B1/6 Ly5.1 mice as
described. (Okada, et al., 1992). Briefly, BM mononuclear cells were isolated
by density
centrifugation over Ficoll Hypaque (<1.077, Pharmacia, Piscataway, NJ).
Lineage
1o negative or low staining cells (fin"°) were obtained by magnetic
bead depletion (anti-rat
immunoglobulin coated beads, Dynal, Oslo, Norway) of mononuclear cells using
the
same lineage cocktail described herein above. The cells were further stained
with
antibodies to Sca-1 and c-kit as described herein above. Sorting for fin
~~°, Sca-1+, and c-
kit cells was accomplished with the Epics 753 as described above.
SfPm cell/stromal cell cocultivation and cytokine-suRp)emented susQension
a r . Stromal cell lines were seeded on tissue culture dishes that had been
coated with
1 % gelatin (Specialty Media, Lavallette, NJ) and were grown at 32°C,
S% COz~ 100%
humidity. Confluent monolayers were irradiated (20 Gy, ' ~~Cesium source,
Gammacell
40, Nordion International Inc. Ontario, Canada) and cultured in modified
Dexter (Dexter,
2o et al., 1984) media (DMEM, 10% FBS, 10% horse serum, 5X10'5 moUL 2-ME, 1X10-
'
mol/L hydrocortisone). For Dexter-LTC, enriched hematopoietic stem cells were
added
and the cultures were maintained at 37° C, S% COz, 100% humidity with
weekly media
changes. The specific numbers of purified stem cells added to stromal cell
cocultures are
given in the appropriate figure or table legends. In some experiments, week 4
AFT024/stem cell cultures were harvested and replated in limiting-dilution
onto fresh,
irradiated (20 Gy) AFT024 monolayers in 96-well trays (Dexter-LTC conditions).
CAs
were scored weekly (as described above), for an additional 5 weeks. Irradiated
(20 Gy)
2018 monolayers in 96-well trays were used in limiting-dilution Whitlock-Witte
assays
(LD-WW) (Whitlock, et al., 1982) to assess stromal-dependent B-lymphopoiesis
content
of both freshly purified and AFT024 cultured fetal liver stem cells. These
cultures were
established in RPMI media with 5% FBS, 2 mmol/L glutamine, 1 mmol/L Na
pyruvate,
and SX10-5 mol/L 2-ME at 37° C, S% COz, 100% humidity. 2018 has been
identified as
a potent B-lymphopoiesis supporting line in W-W conditions. (Deryugina, et
al., 1994).


CA 02340465 2001-02-21
WO 00/11168 60 PCT/US99/19052
Short-term cytokine-supported suspension cultures and short-term AFT024/stem
cell
cocultures were established in Iscove's Modified Dulbecco's Media (IMDM), 10%
FBS,
1% BSA, 5X10-5 mol/L 2-ME at 37°C, S% COZ, 100% humidity. Cytokine
concentrations: rmflk2/flt3-ligand (FL) 30 ng/mL, rmSteel factor (SL) 20
ng/mL, rhIL-6
10 ng/mL. FL was obtained from ImClone Systems Inc.; SL was purchased from
Genzyme Corporation, Cambridge, MA. IL-6 was purchased from Upstate
Biotechnology Inc., Lake Placid, NY.
~ransmantation assavs for hemato~oietic stem cell activity. Competitive
repopulation
was used to measure stem cell activity present in both freshly isolated and
cultured stem
1o cell populations. (Harrison, et al., 1993). This assay was performed using
the congenic
Ly5.1/5.2 mouse system (Morse, et al., 1987). Enriched stem cells were seeded
onto
irradiated stromal monolayers and maintained in Dexter-LTC conditions. At the
end of 4-
7 weeks, the cultures were harvested by vigorous trituration. Single cell
suspensions
were prepared by passage through 22-gauge needles, mixed with fresh congenic
BM and
transplanted into lethally irradiated congenic mice (10 Gy, split dose 3 hours
apart, 1
Gy/min, Gammacell 40). All purified fetal liver stem cells were from Ly5.2
mice. The
competitor BM and recipients were LyS.l. Purified BM cells were from Ly5.1
mice; in
this experiment, Ly5.2 BM was used as competitor and Ly5.2 mice were used as
recipients. In order to assess reconstitution, mice were periodically bled by
capillary
puncture of the orbital venous plexus. Blood (0.1 mL) was collected into
heparin-
containing ( 10 U/mL) DMEM and the red blood cells were lysed with NH,~CI
(Mishell, et
al., 1980). For the experiments described in Figure 15 and Table 4., the
nucleated cells
were divided into two fractions and stained with the appropriate biotinylated
Ly5
antibody and developed with streptavidin-peridinin chlorophyll protein (PerCP)
and; (1)
CD4-FITC, CD8-PE and (2) B220-FITC, Mac-1-PE, Gr-1-PE. Cells from each
fraction
were analyzed on an Epics Profile II, Coulter Electronics. For the experiments
described
in Figure 16 and Tables 2. and 3., nucleated cells were stained with directly
conjugated
lineage antibodies (CD4-PE, CD8-PE, Mac-1-FITC, Gr-1-FITC, and B220-APC) and
biotinylated Ly5.2 antibody which was developed with streptavidin Texas Red
(T.R.).
3o Four color analysis of stained cells was performed on either the Coulter
Epics 753 or
Becton Dickinson FACS Vantage with the appropriate software interfaces
described
above. Anti-Ly5.1 was a kind gift of Dr. H. Nakauchi, University of Tsukuba,
Japan.
Purified and biotinylated Ly5.2 antibody was originally obtained from a
hybridoma


CA 02340465 2001-02-21
WO 00/11168 61 PCTNS99/19052
(AL14A2) kindly provided by Dr. G. Spangrude, University of Utah Medical
Center, Sait
Lake City, UT. In later experiments, the Ly5.2 antibody was purchased from
PharMingen; CD4-PE, CD8-PE, and B220-APC were also purchased from PharMingen.
Streptavidin-PerCP was purchased from Becton Dickinson Immunocytometry
Systems.
Streptavidin T.R. was purchased from Molecular Probes. Competitive
repopulating units
(CRU) per 105 were calculated according to the formula: (Harnson, et al.,
1990).
CRU/105 = %Ly5 positive cells X cell number competitor BM
100-%Ly5 number of test cells
to
Reconstitution values of less than 2% of the test Ly5 donor allele were not
considered
sufficiently above background for calculation of CRU.
Retransplantation potential of stem cells was assessed by secondary
transplantation. Mice from the experiment presented in Figure 14. were
sacrificed 60
weeks after transplantation, BM was harvested and stained with antibody to
Ly5.2
followed by streptavidin T.R. Ly5.2+ cells were collected by cell sorting
(Coulter Epics
753) and used to transplant lethally irradiated secondary Ly5.1 recipients.
Marrow from
mice in the Control and AFT024 groups was used to transplant mice in both
radioprotection and competitive repopulation assays. Ly5.2+ BM from the
primary 2012
2o transplants was used only in radioprotection assays. Primary 2018 mice did
not contain
sufficient Ly5.2+ cells for secondary transplantation. Transplanted mice were
bled and
analyzed by 4-color flow cytometry for the presence of Ly5.2~ cells and
multilineage
reconstitution as described above.
In vitro hemato oip etic progenitor cell assays. The progenitor content of
freshly
purified hematopoietic stem cell populations and AFT024/stem cell cocultures
was
assessed using a variety of in vitro assays. All of the following assays were
accomplished
with fetal liver stem cells enriched as described herein above. To determine
the time
course of CA development, enriched stem cells were seeded onto irradiated
AFT024
monolayers (300-600 cells/well in 12-well trays). CA development was followed
over
3o time and characteristic clusters were quantitated as described above. At
different time
points of the stem cell/AFT024 cultures, individual wells were harvested and
replated into
cytokine-supplemented semisolid clonogenic progenitor assays (CFU-C). The
cytokine-
enriched (rmIL-3 10 ng/mL, rhIL-6 10 ng/mL, rmSL 50 ng/mL, Epo 3 U/mL) methyl-


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cellulose mixture was purchased from Stem Cell Technologies Inc., Vancouver
BC,
Canada. Colonies were scored after 8-14 days of culture at 37°, S% COZ
, 100% humidity
according to established criteria. (Testa, et al., 1993). Colonies that
reached >lmm in
size after 8 days and which contained erythroid bursts and multiple myeloid
cell lineages
including megakaryocytes were scored as high-proliferative potential-mixed
lineage
colonies (CFU-HPP-Mix). (Lowry, et al., 1995). Lineage content of typical
colonies was
determined by Wright's/Giemsa staining of cytospin slide preparation from
individual
colonies. Colony assays were also done with 103 freshly purified cells. The
CFU
progenitor contents of the AFT024 cocultures were normalized to an initial
input of 103
t0 stem cells. To assess the ability of the AFT024 cell line to maintain
primitive lymphoid
progenitors, 4 week cocultures were plated into LD-WW assay on 2018 cells as
described
above. Resulting pro-B cell colonies were scored after 7 days. The cell number
in
individual wells (96-well trays, 8 wells/cell number) was normalized from the
original
number of purified stem cells that initiated the coculture, i.e. stem cell
equivalents/well.
As calculated from the line of best fit, the cell number at 37% negative wells
is the
frequency of pro-B cell colony initiating cells in the starting population.
(Taswell, 1981 ).
In a similar manner, the frequency of CA initiating cells in week 4 stem
cell/AFT024
cocultures was also determined by replating them in limiting-dilution onto
fresh,
irradiated AFT024 monolayers in 96-well trays. CA were scored as described
above, at
1, 2, 3, 4, and 5 weeks after replating in Dexter-LTC. The resulting
frequencies were
calculated as described herein above for the LD-WW assays and are also
expressed in
relationship to the number of stem cells that seeded the initial cultures
(stem cell
equivalents).
Results
Ice, vivo and in vitro assa,Ys f'or stem cell activity maintained by stromal
cell lines.
Highly enriched stem cell populations were used to initiate cultures supported
by single
stromal cell lines. The focus was on one cell line, AFT024, which exhibited
particularly
potent stem/progenitor cell support. Two other stromal cell lines, 2012 and
2018,
(Wineman, et al., 1996) were included in some experiments. In order to more
rigorously
3o establish the clonality of these lines, they were subcloned by limiting-
dilution. All
subclones obtained from a given cell line contained the same proviral
integrant position
as the parental cell line. The AFT024 cell line was evaluated both for its
ability to
maintain in vivo competitive repopulating stem cells, as well as a broad
spectrum of


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stem/progenitor cells defined by a variety of in vitro assays. The in vivo
assays focused
on the ability of stem cells, cultured for extended time periods (4-7 weeks),
to
permanently reconstitute mufti-lineage hematopoiesis in transplanted hosts.
The in vitro
assays included the enumeration of CA appearing over time in the initial
cultures as well
as the quantitation of stemlprogenitor cells which can form colonies in
cytokine-
supplemented replating assays. Cells from four-week AFT024/stem cell
cocultures were
also assayed by limiting-dilution for the content of progenitors capable of
initiating
secondary CAs on AFT024 or B-lymphopoiesis in Whitlock-Witte cultures
supported by
2018.
to AFT024 maintains auantitative levels of long-term in vivo repo~u'ating stem
cell
'vi . One line of investigation inquired if and at what levels in vivo
transplantable
stem cell activity was present in four to seven week-old cultures initiated
with highly
enriched stem cells and supported by AFT024, 2012 or 2018. Purified day 14
fetal liver
cells (AA4.1+, liri ~~°, Sca-1+, c-kit+) and adult BM cells
(lin'~~°, Sca-I+, c-kit+) were used as
t5 sources of stem cell activity. Both of these populations are about 1000 to
1500-fold
enriched for stem cell activity, as measured by competitive repopulation.
(Harrison, et al.,
1993). The LyS.I/Ly5.2 congenic system was utilized for all competitive
repopulation
studies. (Morse, et al., 1987). The data presented in Figure 14 demonstrate
that the
cultures supported by AFT024 contain stem cell activity at levels
quantitatively identical
2o to those present in the uncultured purified populations. In this
experiment, individual
Ly5.1 mice received 103 freshly purified Ly5.2 cells or the cultured
equivalent of 103
purified Ly5.2 cells. Each mouse also received 10~' LyS.I competitor BM cells.
The
percentage of Ly5.2 positive peripheral blood cells was approximately equal in
both
groups of recipient animals. Moreover, the cultured stem cell activity is as
effective as
25 freshly purified activity for in vivo periods of greater than one year. The
data in Figure 14
also show that the 2018 cell line is completely ineffective in maintaining
highly purified
stem cell activity while the 2012 cell line supports intermediate levels of
repopulating
activity. The data presented in Table 4A. provide quantitative competitive
repopulating
unit (CRU) value calculations as well as the results of multiparameter lineage
analyses.
3o The extremely low levels of reconstitution by 2018-cultured stem cells
precluded lineage
analysis. The CRU values of the AFT024-cultured and freshly purified
populations are
nearly identical. Moreover, both fresh and AFT024-cultured stem cells
reconstitute
myeloid and lymphoid cell populations to a similar degree. In order to further
access the


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supporting activities of AFT024 and 2012 we utilized a 10-fold lower number of
fetal
liver stem cells from two separate purifications to initiate the cocultures.
The cultures
were continued for 4-7 weeks, harvested, and used in competitive repopulation
studies.
Each recipient received the cultured equivalent of 100 purified Ly5.2 stem
cells plus
4X105 Ly5.1 competitor BM cells. A total of twelve mice were transplanted with
AFT024 cocultures ( four each aver four, five and seven weeks of LTC). The
parental
AFT024 line was used in the 4 week group and two different subclones were used
to
support the five and seven week cultures. The AFT024-cultured Ly5.2 stem cells
contributed to 20-30% of peripheral blood cells in these recipients while
cells cultured on
l0 2012 demonstrated more limited in vivo function (Table 4B). The 2012
cultures were
done with two subclones of the parental line and were maintained for four
weeks prior to
harvest and transplant (four mice/subclone). The data utilizing different
cultures time or
sublcones did not vary significantly from each other and are presented
together in Table
4B. An additional experiment was undertaken using enriched BM which was
cultured on
AFT024 and 2018 for six weeks. In this experiment, BM was purified from Ly5.1
congenic mice. Each Ly5.2 recipient mouse in this study received 100 freshly
purified
cells, or the cultured equivalent of 100 purified cells. Both groups received
105 Ly5.1
competitor BM cells per mouse. Data analysis, for the presence of LyS.I+ cells
at 4
months after transplant, is presented in Table 4C. For 6 weeks of culture,
AFT024 cells
maintained quantitative levels of reconstituting activity present in 100
purified BM stem
cells. The 2018 cell line failed to maintain stem cell activity.
These studies were extended to include secondary transplantation as an
additional
assay for primitive stem cells. BM cells were harvested from the primary
recipients of
fresh and cultured fetal liver stem cells (see Figure 14. and Table 4A.) and
the Ly5.2
positive, fetal liver-derived fraction was collected by cell-sorting.
Secondary
radioprotection and competitive repopulation transplants were performed. The
data are
presented in Table S. The secondary recipient repopulating activities are
nearly identical
for the AFT024 cultured stem cells and the non-cultured controls. Lineage
analysis of the
Ly5.2 cells in the secondary recipients revealed similar numbers of myeloid
and
lymphoid cells derived from both AFT024-cultured and non-cultured stem cells.
Some
level of secondary reconstituting cell activity was observed.
Additional experiments were also performed to determine where the levels of
stem cell activity present in long-term AFT024 cocultures were compared to
those present


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in short-term cytokine stimulated cultures or in short-term AFT024-supported
cultures
(Figure 15). Purified fetal liver cells were seeded onto an AFT024 monolayer
and
maintained in Dexter-LTC conditions for 5 weeks. Simultaneously, the same
numbers of
purified cells were cultured for 5 days with; ( 1 ) different cytokine
combinations or (2) on
AFT024. The transplantable activity in the cultured cells was then assayed by
competitive repopulation. Each mouse received the cultured equivalent of 600
stem cells
together with 4X 105 LyS.I competitor BM cells. It is evident from the data
that the levels
of in vivo repopulating activity present in the long-term AFT024-supported
cultures are
much greater than those remaining after a short-term cytokine-supported
culture period.
to Of interest also is that short-term AFT024 stem cell cocultures do not
maintain significant
in vivo reconstituting activity. In fact, these levels of stem cell activity
are identical to
the levels seen in the cytokine-supported cultures.
In vitro stemlprogenitor populations are expanded by AFT024. AFT024/stem cell
cocultures have vigorous hematopoiesis throughout the entire in vitro culture
period.
is This is reflected in the large numbers of relatively mature hematopoietic
cells which are
produced throughout the culture period. In addition, CA colonies are observed
throughout the culture period. Figure 16 shows a time course of CA appearance
with
purified fetal liver stem cells (3 separate experiments). After 28 days in
culture,
approximately one in every twenty input stem cells is capable of proliferating
into a CA.
2o In addition, CA appearance over time follows a biphasic distribution, with
many CA
observed early in the culture period. In order to enumerate the various
classes of
stem/progenitor cells present in AFT024 cocultures, we performed a series of
in vitro
replating experiments. These included the quantitation of: (l.) progenitor
cells capable
of colony-formation in cytokine-supplemented semisolid cultures (CFU assay),
(2.)
zs progenitor cells capable of initiating secondary CA in limiting-dilution
AFT024 cultures
and (3.) progenitor cells which can initiate B-lymphopoiesis in LD-WW
cultures. In all
of these experiments the primary cultures were initiated with purified fetal
liver cells. All
data presented below are normalized to an initial input of 103 purified cells
(CFU assay)
or the actual number of initial input stem cells (limiting-dilution assays).
3o Shown in Figure 17 are the numbers and types of cytokine responsive CFU
progenitors present at various times in the AFT024-supported cocultures.
Production of
CFU is evident at all time points. However, the content is especially high
after four
weeks, representing a 5-7 fold increase/expansion when compared to the content
in the


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freshly purified populations. The content of more primitive progenitors (CFU-
HPP-Mix)
is increased by 12-fold. These HPP-Mix colonies often reach a size of 2 mm in
8 days
and contain large numbers of erythroid bursts and megakaryocytes.
Interestingly, there
does not appear to be a correlation between CA number and CFU content at
different
culture times. This is most apparent at day 6, when CA numbers are at their
peak but the
progenitor content is similar to that observed in non-cultured stem cells.
Furthermore,
there is no correlation between CFU content and the absolute numbers of
maturing
hematopoietic cells present in a given culture.
Next, the content of primitive B-lymphoid progenitors present in the
1o AFT024/stem cell cultures was determined. This was accomplished by plating
cells from
the four-week AFT024 cocuitures into LD-WW assays over the 2018 stromal cell
line.
Two experiments with freshly purified stem cells and AFT024-cultured stem
cells
showed that the frequency of pro-B cell progenitors is expanded 10-fold in
AFT024
cultures compared to the frequency observed in the freshly purified input
population (day
0 frequency 1 in I 1.0, rZ= 0.98; day 28 AFT024-cultured frequency 1 in 1.1,
rz= 0.97).
In order to measure the content or frequency of progenitor cells capable of
initiating secondary CA, 4 separate, four-week AFT024/stem cell cocultures
were
replated in limiting-dilution onto fresh AFT024 monolayers. CAs were scored
after one
week. The data are presented in Figure 18A. Large numbers of secondary CAs
were
observed. When normalized to the stem cell numbers used to initiate the
primary cultures
(stem cell equivalents, see Methods), the frequency of these progenitors is 1
in 3 to 4.
Figure 18B. shows data from one of the 4 experiments presented in Figure 18A.,
where
the quantitation of secondary CAs was extended for 4 more weeks. The frequency
of CA
decreases slowly over time (1 in 19 after an additional 4 weeks),
approximating the
frequency seen in the primary cultures at four weeks. In summary, our in vitro
replating
assays collectively demonstrate a significant expansion of primitive
progenitor
populations in 4 week AFT024 cultures. In these same cultures there is no
decrease in the
levels of transplantable stem cell activity present in the total hematopoietic
cell
population.
Discussion
In this example, it was demonstrated, that the AFT024 stromal cell line can
maintain quantitative levels of in vivo repopulating stem cells for at least 7
weeks of in
vitro culture. Highly enriched stem cell populations in low numbers ( 100
cells) were


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used and cell activity was measured in a stringent, competitive repopulation
assay
system. The cultured stem cell activity satisfies all in vivo criteria
normally ascribed to
the most primitive stem cell compartment; (1) long-term engraftment ability,
(2)
multilineage potential and (3) the ability to repopulate secondary recipients.
In addition,
the studies with the low culture initiating stem cell numbers for both BM and
fetal liver,
imply that AFT024 stromal cells exert their supportive effects in a direct
manner. These
studies represent a clear example of an in vitro system capable of directly
supporting the
most primitive stem cell compartment.
Other data revealed that stromal cell lines isolated from a single tissue
source are
to heterogeneous with respect to their abilities in maintaining long-term
repopulating stem
cells. (Wineman, et al., 1996). It was speculated that the rare cell lines
which were
effective in supporting in vivo reconstituting stem cells may represent
immortalized
components of in vivo stem cell niches. However, the data argue for the
necessity of
using purified stem cell populations in order to support such a hypothesis.
Specifically,
the 2018 cell line maintained transiently reconstituting activity present in
unfractionated
BM. However, 2018 fails to maintain measurable repopulating activity when
cultured for
4-6 weeks with highly purified BM or fetal liver stem cells. Similarly, in
experiments
using purified cells, there was a failure to show robust levels of
reconstituting stem cell
activity in cultures supported by CFC034, the most effective cell line in the
whole BM
2o studies (Wineman, et al., 1996). The 2012 cell line which was reported
(Wineman, et al.,
1996) to be effective in maintaining long-term repopulating whole BM derived
stem cells
is only partially effective in the present studies. Moreover, only some
subclones of 2012
display such activity (in spite of identical proviral integration positions in
all subclones).
Recently, studies have shown that the S 17 cell line which consistently
supports the stem
cell activity present in whole BM, (Wineman, et al.,. 1993) is not similarly
effective in the
maintenance of purified BM stem cells. (Szilvassy, et al., 1996). Taken
together with
these current data, the previously observed stem cell supporting stromal cell
activities
may reflect the actions) of indirect mechanisms and therefore do not permit
the
identification of cellular stem cell niche components. One previous study has
shown that
3o the Sysl stromal cell line can maintain high levels of transplantable
activity present in
purified BM. (Szilvassy, et al., 1996). The competitor cells in that study
were
compromised by prior serial transplantation. Moreover, the culture period was
extended
for only two weeks and effective maintenance required the addition of
exogenous


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leukemia inhibitory factor. In contrast, AFT024 is a cell line that provides a
direct-acting
long-term stem cell supporting environment without the addition of exogenous
factors.
In addition to recovering net input levels of transplantable activity from
AFT024
supported cultures, significantly expanded populations of primitive progenitor
cells were
also obtained. CFU-HPP-Mix progenitors are expanded by 12-fold after 4 weeks
of
culture and the numbers of stromal-dependent pro-B lymphoid progenitors are
similarly
amplified. This suggests that the AFT024-mediated process of stem cell
maintenance is
in reality a dynamic phenomenon. Specifically, during the first portion of the
culture
period, the majority of transplantable stem cell activity may be lost, through
l0 differentiation or cell death. The remaining primitive stem cells may
expand to yield
input levels of transplantable activity as well as increases in the numbers of
more
committed progenitors. One hypothesis is that short-term AFT024 supported
cultures
should contain reduced levels of transplantable stem cell activity. Figure 17
support this
hypothesis. The standard Dexter-type media used in parallel long-term AFT024
cocultures was not used in these short-term cultures. However, in another
short-term
experiment, utilizing Dexter-LTC media, a similarly dramatic reduction in stem
cell
activity was observed after 4 days of culture on AFT024.63 These observations
are
intriguing because they suggest that the AFT024 cell line is able to
facilitate some degree
of ex vivo transplantable stem cell proliferation and expansion. Indeed, in
other studies it
2o was shown that AFT024 can support colony formation initiated by single
purified stem
cells with B and T-lymphoid, myeloid and erythroid potentials. Moreover, the
data
suggest that is possible to efficiently introduce retroviral markers into
transplantable stem
cells at various times during AFT024 cocultures. Extension of such marking
experiments
and an analysis of proviral integration patterns will be necessary to
rigorously ascertain if
self renewal replication is occurring during these coculture periods.
The ability of AFT024 to maintain the most primitive stem cell compartment
while generating and expanding at least some less primitive members of the
stem/progenitor cell hierarchy raises interesting issues regarding the nature
of stem cell
niches. The present invention suggests that microenvironmental niche models
which
3o postulate distinct cellular entities responsible for stem cell self renewal
and other cellular
entities which support the generation of committed progenitor cells may be
overly
simplified. (Uchida, et al., 1993). Quite clearly a single microenvironmental
cell type
represented by AFT024 is sufficient for keeping stem cells in an
undifferentiated state as


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well as allowing commitment and progenitor expansion to take place. A hallmark
feature
of a stem cell niche is the ability to facilitate the generation of the entire
stem/progenitor
cell hierarchy from very primitive cells. Therefore, the main functional role
of such
niches may be to provide an environment which permits the production of the
correct
numerical balance of more and less primitive stem/progenitor cell entities.
This model
contains several testable hypotheses. The most important is that in vitro stem
cell
maintenance should not be interpreted, literally, as the maintenance of
quiescent cells but
rather as a phenomenon which results from a balance of self renewal and
commitment
decisions which occur during stem cell division.
1o A cytokine cocktail of IL-6, SL, and FL is not effective in maintaining
fetal liver
stem cell activity. It was shown that RNA transcripts for these and 10 other
cytokines are
present in AFT024, but they are also detected at similar levels in non-
supporting lines
such as 2018. (Wineman, et al., 1996). These observations suggest the
existence of novel
AFT024-derived molecules which may act on stem cells. Indeed, using a
subtractive
hybridization molecular cloning strategy, a number of candidate molecules have
been
identified. Two of these molecules contain EGF-like repeat motifs that are
most closely
related to those found in the Notch/Notch-ligand family. Interestingly, one of
these
molecules appears to have activity on primitive stem cell populations. (Moore,
et al.,
1997). (See, Examples presented herein above.
2o Table 4. Multilineage stem cell activity, high CRU levels maintained in
AFT024 cultures
A. With 1000 purified fetal liver cells
Lineage Contribution, % Ly 5.2 Cells Total
Cells Week CD4 CDS B220 Myeloid % Ly 5.2 CRU/105
Control 5 3.3+1.5 4.4~1.7 48+5.1 32+6.0 28~1.3 3$6~24
12 27~10 24~8.7 63~8.9 43+9.2 44~3.2 800~102
24 42+6.9 34+4.1 71+4.4 62+1.4 48+4.8 984+171
AFT024 5 7.7~6.2 6.0~3.5 58~6.6 36+4.7 39~2.1 641~54
12 44~7.1 38+6.4 70~5.6 53~3.6 48+1.4 942+51
24 56+3.8 49+3.1 722.4 643.7 561.9 1270+96


2012 5 0.8+0.5 1.40.3 6.8+3.6 1411 7.4+2.3 528.5


12 9.1 $.4 106.9 149.3 23+20 14+4.5 19468


24 173.8 182.0 235.9 326.4 21+4.4 19671




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B. With 100 purified fetal liver cells
Lineage Contribution, y5.2 CellsTotal
% L


Cells Week GranulocytesB-cells T-Cells % Ly5.2 CRU/105



AFT024 15 334.6 416.0 255.3 284.6 1888315


26 17+4.4 18+4.8 185.9 245.9 1825+571


46 22+5.7 19+6.6 15+6.7 20+6.5 1595628


2012 15 32+7.8 28+7.2 205.2 24+6.6 1390+382


26 102.8 9.7+3.2 9.73.2 143.9 699176


46 11+2.3 6.7+1.6 8.93.2 9.02.0 409+
80


C. With 100 purified bone marrow cells
No. of Lineage Contribution, % Ly 5.1 Cells Total
Cells mice CD4 CD8 B220 Myeloid % Ly 5.1 CRU/105
Control 8 38~7.3 30~5.8 43+6.7 31~4.8 32+6.5 583+172
AFT024 7 20~9.8 18+8.8 20~8.7 24~11 22~10 490~296
A. AA4.1 day 14 fetal liver cells (Ly 5.2) were further purified for a lin' ',
Sca-1 , ckit stem cell surface
phenotype. Fresh purified control cells, 103, were transplanted with 10~'
Ly5.1 competitor marrow (n=6
4
mice). From the same purification, 10 cells were cocultured with stroma! cell
lines for 4 weeks.
Subsequently, 10% of each culture was transplanted per mouse (n=8 mice/stroma)
together with 10''
competitor BM cells. The contribution to each lineage in peripheral blood is
expressed as the percent of the
total specific lineage population that was Ly5.2'. CRU/10', relative
enrichment of competitive
repopulating units. Data are presented +SEM.
B. The lineage and CRU content of low numbers of enriched fetal liver stem
cells maintained on AFT024
and 2012 were determined. 500 stem cells were maintained in Dexter-LTC for 4-7
weeks over irradiated
monolayers. 20% of each culture was used to transplant groups of 4 mice (i.e.
each mouse received the
equivalent of 100 stem cells that initially seeded the cultures) combined with
4X105 competitor BM cells.
Data are presented as ~SEM.
C. Ly 5.1 BM cells with a lin'~°, Sca-1+, and c-kit' cell surface
phenotype were purified. 100 fresh cells
per mouse (Control) were transplanted with 105 Ly 5.2 competitor BM cells. One
thousand of the same
purified cells were cocultured with stromal cell lines for 6 weeks. The
cultures were then harvested and
10% of each culture was transplanted per mouse with competitor. Data are from
peripheral blood samples
taken 4 months after transplant and are presented +SEM.


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Table 5. LTRSC maintained on AFT024 are able to repopulate secondary
recipients at
levels comparable to non-cultured stem cells.
Ly 5.2T peripheral blood cells


Group Weeks Radioprotection Comp.


Repopulation


Control 6 21 (1) 2.60.6 (4)


22 54 ( 1 ) 5.61.6 (4)


AFT024 6 130.2 (4) 1.3+0.9 (8)


22 4415.I (3) 4.30.9 (8)


2012 6 6.72.9 (4) ND


22 142.2 (4) ND


The retransplantation potential of LTRSC in primary recipients of stromal cell
cultured
stem cells was studied in secondary recipients. 60 weeks after transplant,
primary mice
(see Figure 14, Table 4A.) were sacrificed, BM harvested and stained with
antibody to
Ly5.2. Ly5.2+ cells were collected by cell sorting and used to transplant
secondary
recipients (congenic LyS.I mice). Control and AFT024 groups were transplanted
with
1.5X106 Ly5.2 cells/mouse for radioprotection (4 mice/group were transplanted)
and
to 7.5X105 Ly5.2 cells + 7.5X105 Ly5.1 cells for competitive repopulation (4
mice for the
Control group and 8 mice for the AFT024 group). 2012 mice were transplanted
with
3X105 Ly5.2 cells/mouse (4 mice). Weeks are the times after transplant that
the mice
were analyzed. (n), number of mice surviving/group; ND, not done. Data are
presented
+SEM.
EXAMPLE 4: Hematopoietic activity of a stromal cell transmembrane protein
containing epidermal growth factor-like repeat motifs
Primitive hematopoietic stem cells are closely associated with discrete in
vivo
microenvironments. These "niches" are thought to provide the molecular signals
that
2o mediate stern cell differentiation and self renewal. The fetal liver
microenvironment was
dissected into distinct cellular components by establishing an extensive panel
of stromal cell
lines. One particular cell line maintains repopulating stem cells for
prolonged in vitro
culture periods. A subtraction cloning strategy has yielded a cDNA which
encodes a cell
surface glycoprotein with a restricted pattern of expression among stromal
cell lines. This


CA 02340465 2001-02-21
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molecule, previously identified as delta-like/pre-adipocyte factor-1, contains
epidermal
growth factor-like repeats which are related to those in the
notch/delta/serrate family of
proteins. The potential role of this molecule in hematopoietic stem/progenitor
cell regulation
was investigated. The delta-like protein displays activity on purified stem
cells by promoting
the formation of "cobblestone areas" of proliferation. These cobblestone areas
contain both
primitive high-proliferative potential progenitors and in vivo repopulating
stem cells.
Introduction
The positive and negative regulatory mechanisms that govern the proliferation,
self
renewal and differentiation of primitive hematopoietic stem cells are complex
and poorly
understood ( Ogawa, M. (1993) Blood 81, 2844-2853). Numerous cytokines have
been identified
that, when used iu vitro appear to act directly on purified stem cells by
promoting
proliferation and differentiation. However, attempts to demonstrate the in
vitro maintenance
and/or expansion of transplantable pluripotent stem cells using defined
cytokine
combinations have been largely unsuccessful (Knobel, K. M., et al., 1994;
Peters, S. O., et
al., 1995). Moreover, it is unclear to what extent any currently identified
cytokines reflect
mechanisms that are responsible for regulating normal, in vivo, stem cell
behaviors. It is
widely accepted that in vivo, stem cells are intimately associated with
discrete
microenvironmental "niches" (Wolf, N. S. 1979). Such niches are likely sources
for the
molecular signals which collectively mediate the differentiation and self
renewal of stem
2o cells. Indeed, it has long been possible to demonstrate that preestablished
stromal cell
monolayers derived from hematopoietic tissues can support long-term
hematopoiesis in vitro
( Dexter, T. M., Allen, T. D. & Lajtha, L. G. 1977). The long-term nature of
these cultures,
together with the continuous production of committed progenitor cells suggest
that both self
renewal and commitment decisions can occur in vitro. At the cellular level.
the
hematopoietic microenvironment consists of numerous distinct cell types.
Previous studies
have shown that this cellular heterogeneity reflects a similarly broad
heterogeneity in terms
of hematopoietic supportive abilities (Deryugina, E. L, et al., 1994). Some
cloned stromal
cell lines can support stem cell activity in vitro, while others are
ineffective. Similarly,
distinct stromal cell types appear to influence the outcomes of stem cell
differentiation
3o processes (Friedrich, C., et al., 1996). Recent studies have shown that
stromal cell lines that
efficiently maintain long-term transplantable stem cells in vitro for
prolonged intervals
represent a small fraction of the total stromal cell population ( Wineman, J.,
et al., 1996). A
fetal liver stromal cell line, AFT024 was identified which maintains high
levels of


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transplantable multilineage stem cell activity for extended in vitro culture
periods (Moore,
K. A., Ema, H. & Lemischka, I. R. (1997) (See, Example 3). The stem cells used
to initiate
these cultures are highly purified. It is, therefore, likely that in this
system the mechanisms
that mediate stem cell maintenance do so by acting directly on primitive stem
cells. A
s different fetal liver-derived cell line, 2018, fails to maintain long-term
repopulating stem cell
activity. A PCR-based RNA expression analysis of 13 cytokines reveals
qualitatively
identical expression patterns in AFT024 and 2018. It wastherefore,
hypothesized that the
hematopoietic supportive ability of AFT024 is, at least in part, mediated by
novel gene
products not expressed in 2018.
to Using a subtractive hybridization approach a number of cDNA clones were
identified
which were specifically expressed in AFT024 but not in 2018. The sequence of
one cDNA
was identical to a molecule that encodes a transmembrane protein that contains
six epidermal
growth factor (EGF) repeat motifs. The EGF-like repeat sequences of this
molecule,
variously known as delta-like (dlk) ( Laborda, J., et al., 1993), preadipocyte
factor-1 (Smas,
is C. M. & Sul, H. S, 1993), and stromal cell protein-1 (Genbank, D16847), are
most closely
related to those present in the notch/delta/serrate family of signaling
proteins. In Drosophila
and Cuenorhabclitis, these molecules are required for correct cell-fate
specification decisions
in a variety of tissues (Rebay, L, et al., 1991 ). Vertebrate homologs of the
notch/deltalserrate family have been identified (Ellisen, L. W., et al., 1991;
Bettenhausen, B.,
2o et al., 1995; Lindsell, C. E., et a1.,1995). While the exact functional
relationship of dlk to the
activities of this family of molecules is unclear, in one in vitro study, it
has been shown to
block adipocyte differentiation ( Smas, C. M. & Sul, H. S, 1993). No studies
have been
reported that demonstrate a hematopoietic function for dlk. Expression
analyses and these
observations show a limited temporal pattern of dlk expression during murine
fetal
2s development which coincides with the time period of hematopoietic stem cell
expansion
(Smas, C. M. & Sul, H. S, 1993).
Functional studies were undertaken to determine if dlk can act as a
hematopoietic
regulator. This molecule affects highly enriched stem cell populations by
promoting
"cobblestone area" (CSA) colony formation in dexter-type stromal cocultures.
These CSA
3o colonies contain an expanded population of primitive, high proliferative
potential myeloid-
erythroid progenitors. These cultures also contain stem cells capable of in
vivo engraftment
at levels equivalent to those present in parallel AFT024 supported cultures.
It is proposed
that dlk represents one molecular component responsible for the hematopoietic
supportive


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ability of AFT024. As such, dlk may define a novel molecular pathway of stem
cell
regulation by the microenvironment.
Materials and Methods
Cell Lines and Culture. The fetal liver stromal cell lines used in this study
were
derived as previously described (Wineman, J., et al., 1996). Cells were
routinely cultured in
DMEM containing 10% fetal bovine serum (FBS) and 50 uM b-mercaptoethanol (2-
ME),
and maintained at 31°-33°C, S% CO2. For long-term cocultures
with hematopoietic stem
cells, confluent monolayers were irradiated (20 Gy), placed in modified Dexter
media
(DMEM, 10% FBS, 10% horse serum, 50 uM 2-ME, 0.1 uM hydrocortisone) and
l0 maintained at 37° C, S% COZ with weekly media changes. NIH3T3 cells
were obtained from
ATCC.
d(k Expression Ana~rsis. Total RNAs from stromal cell lines were poly A+
selected,
Northern blotted, and hybridized to 'ZP-labeled probes according to standard
protocols
(Sambrook, J., Fritsch, E. F. & Maniatis, T, 1989). A 600 by dlk-cDNA clone
from the
t 5 AFT024 subtracted library was used as a probe. cDNA templates for RT-PCR
were
prepared according to manufacturers' protocols (GIBCO/BRL). Oligonucleotide
primers
were: sense 5'- GACCCAGGCTGCCCC-3' (SEQ.ID.No.:85) and antisense 5'-
GGTACTCTTGTTGAG-3' (SEQ.ID.No.:86). For analysis of dlk expression at the
protein
level, antisera specific for dlk was generated by immunizing rabbits with a
Flag-dlk fusion
2o protein (described below). Resultant antibodies were purified by affinity
chromatography.
Ceil surface expression of dlk in stromal cell lines was accomplished by flow
cytometry.
Cells were incubated with dlk antibody and a similarly prepared irrelevant
control antibody.
Specific labeling was developed by donkey anti-rabbit-fluorescein
isothiocyanate (Jackson
ImmunoResearch). Stained cells were analyzed on a Becton Dickinson FACScan
using Cell
25 Quest software.
dlk Fusion Protein Preparation. The expression plasmid pCD4-Ig contains cDNA
for
the extracellular domain of human CD4 fused to genomic sequences of the human
immunoglobin heavy chain (Zettlmeissl, G., et al., (1990). cDNA for CH2-CH3 of
human
IgG~ (Goodwin, R. G., et al., 1990) was cloned into EcoRI and NotI sites of
pcDNA3
30 (Invitrogen) to give the plasmid KB52.3.2. cDNA encoding the extracellular
domain of dlk
was obtained by RT-PCR with primers BP 151 and BP 152 using total RNA from NIH
3T3
cells as template. The resulting PCR fragment was cloned into KB52.3.2 via
HindIII and


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EcoRI sites to obtain the soluble dlk-Ig expression plasmid. pdlk-Ig or pCD4-
Ig were
transfected into NIH3T3 cells together with pSVNeo and stable clones were
isolated.
Soluble CD4-Ig and dlk-Ig fusion proteins were harvested and then purified by
affinity
chromatography on HiTrap Protein G-sepharose (Pharmacia). Primers: sense BP 1
S 1,
5'GAGGGTACCAAGCTTCGTGGTCCGCAACCAGAAG-3' (SEQ.ID.No.: 87);
anti-sense BP 152, S'-CTCAGATCTGAATTCGGCCTGTCCCTCGGTGAGGAG-3'
(SEQ.ID.No.: 88).
Flag-dlk fusion protein was used to immunize rabbits for the production of dlk
antiserum. The protein expression plasmid pcDNA3-Flag is a modif cation of the
plasmid
1o pcDNA3 (Invitrogen) and contains the coding region for the Flag peptide
(DYKDDDDKI)
{Hopp, T. P., et al., 1988) as well as a BgIII restriction site. A cDNA
fragment encoding the
extracellular domain of dlk was obtained by RT-PCR using RNA from NIH3T3
cells.
Primers: sense BP 155,
5'-GACAAGATCTCAGCTGAATAGCGACCCACCCTGTG-3' (SEQ.ID.No.: 89);
antisense BP 154,
S'-GCATCTAGAGCGGCCGCTCAGGCCTGTCCCTCGGTGAGGAG-3' (SEQ.ID.No.:
90). The PCR fragment was ligated into pcDNA3-Flag to yield pFlag-dlk. pFlag-
dlk was
transfected into cos cells. Purification of the Flag-dlk protein from cos-
conditioned media
was performed according to manufacturer's directions using the Flag monoclonal
antibody,
M 1, immobilized on agarose (International Biotechnologies).
gJ'asmid Constructs and Table Transfection. Full-length murine dlk cDNA was
obtained by RT-PCR with primers BP 1 S 1 (see above) and antisense BP 200:
5'GCATCTAGAGCGGCCGCGAACGCTGCTTAGATCTCCT-3' (SEQ.ID.No.:91), using
total RNA from NIH3T3 cells as template. The product was subcloned into the
vector
pCRII (Invitrogen) and then cloned into a retroviral expression vector
(Kitamura, T., et al.,
1995), (G. Nolan, Stanford University), via the primer-encoded HindIII and
NotI sites.
Supercoiled plasmid was transfected into BFC012 stromal cells together with
the pZeo
(Invitrogen) selectable marker and selected in 50 ug/ml Zeocin (Invitrogen).
BFC012 cells
also were transfected with pZeo alone and selected as above. Clones from both
selected
3o populations were isolated and all remaining colonies ( 100-200 per dish)
were pooled and
expanded as populations.
Hematonoietic Stem Cells and In yitro Hematopoiet~'r Away. Hematopoietic stem
cell populations were derived from wild type, Ly5.2-C57B1/6J (Jackson
Laboratories), day


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14 fetal liver, enriched for the AA4.1~, Sca-1+, c-kit+, and line°~-
phenotype, by
immunopanning and fluorescence-activated cell sorting as described (Jordan, C.
T., et al.,
1995). Adult hone marrow (BM) was used directly after density centrifugation
and
immunomagnetic bead depletion or was further enriched for Sca-1+, c-kit+,
line°~~ cells by
flow cytometry as described (Okada, S., et al., 1992). Cell sorting and data
analysis was
accomplished with a Becton Dickinson FACS Vantage using Celt Quest software.
Stromal
cell/stem cell cocultures were initiated in 12-well trays with 300-1,000
enriched stem cells
per well. Cobblestone areas were quantitated by inverted-phase microscopy as
described
(Ploemacher, R. E., et al. , 1991 ). Clonogenic progenitor assays were
performed with either
freshly purified stem cells or cells harvested from the stromal cocultures.
These were
cultured in cytokine-containing semisolid media according to the
manufacturer's
recommendations (Stem Cell Technologies, Vancouver, BC). Soluble dik and
control fusion
proteins were added to semisolid progenitor assays at concentrations of 0.1,
0.5 and 1.0
ug/ml and also to BFC012 stromal cocuItures at concentrations of 0.1 ug/ml.
Fusion protein
was replenished weekly in the stromal cocultures.
Compgljtive repopulating tra~,~t~lantation assay. Cultured cells were
harvested,
combined with fresh unfractionated BM obtained from congenic C5'7B1/G Ly5.1
mice
(National Cancer Institute) and transplanted into lethally irradiated ( 10 Gy,
split dose 3 h
apart from a ~3'Cs source, 1 Gy/min) Ly5.1 recipient mice. Each mouse received
2 X 105
2o competitor BM cells and a fraction of the cocultured stem cells. Mice were
bled by capillary
puncture of the orbital venous plexus and 100 ul was collected; red blood
cells were
removed by NHaCI lysis. The nucleated cells were stained for the Ly5.2
(CD45.2) allelic
marker using either fluorescein isothiocyanate-labeled directly conjugated
Ly5.2 monoclonal
antibody or a biotinylated form developed with streptavidin conjugated to
Texas red. Cells
also were stained with directly conjugated antibodies to lineage markers. All
antibodies and
chromogens were obtained from Pharmingen. Flow cytometric analysis was done on
a
Becton Dickinson FACS Vantage using Cell Quest software.
Results
Genes expressed in AFT024 but not in 2018 were identified by a subtractive
cloning
approach. Sequence analysis identified one of these AFT024-specific clones as
dlk.
Expression studies, (Figure 19A) show high levels of dlk in AFT024 and
subclones isolated
from this line, but undetectable levels in 2018 and BFC012. The latter two
stromal cell lines


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do not support repopulating stem cells. The PA6 stromal cell line and NIH
Swiss 3T3 cells
both show expression of dlk and were the cell sources for identification of
SCP-1 and dlk,
respectively. PA6 cells have been shown to support in vitro hematopoiesis and
long-term, in
vivo repopulating stem cells (Kodama, H., et al., 1992). Swiss 3T3 cells are
also capable of
supporting multipotent hematopoietic stem cells in vitro, promoting CSAs and
maintaining
in vivo spleen colony-forming units (CFU-S) (Roberts, R. A., et al., 1987).
Interestingly, an
additional fetal liver stromal cell line, 2012, which has some degree of stem
cell supporting
activity (Wineman, J., et al., 1996), and its subclones also express dlk.
Furthermore, an RT-
PCR analysis (40 cycles) of an additional 10 fetal liver-derived stromal cell
lines and several
t0 other lines, shows detectable levels of d1k in only two additional lines
(Figure 19B). These
two cell lines (CFC032 and CFC008) can maintain some level of long-term
transplantable
stem cell activity present in whole BM (Wineman, J., et al., 1996). A
correlation between a
stromal cell line's ability to support stem cells and the expression of dlk
was suggested.
Therefore, functional studies were undertaken in order to delineate if dlk can
act on or
t 5 modulate hematopoietic stem cells.
Soluble dlk protein was added to progenitor cultures in semi-solid media. The
soluble
protein consisted of the dlk extracellular domain fused to the Fc portion of
human IgG, . The
stem cell sources in these assays were highly enriched fetal liver cells
(AA4.1+, lin'°~-, Sca-
1+, c-kit +). The influence of soluble dlk on hematopoietic progenitor colony-
formation was
zo assessed. As shown in Table 6, no differences were noted either in the
number, sizes, or
lineage compositions of colonies. Identical results were obtained at dlk
concentrations
ranging from 0.1 to 1.0 ug/ml. In addition, no differences were noted in
similar studies
using enriched BM cells (Sca-1+, c-kilt, lin"°).
Evidence for a positive effect of the dlk protein on stem/progenitor cells was
25 observed when the soluble form was added to dexter-type cocultures. For
these studies a
stromal cell line (BFC012) was used that neither expresses endogenous dlk (see
Figure 19)
nor maintains significant in vitro hematopoiesis. In four experiments, two
each using highly
enriched adult BM and fetal liver stem cells, we monitored the appearance of
CSAs over
time. These colonies provide a convenient, quantitative estimate of
hematopoietic activity
30 initiated by primitive stromal dependent stem/progenitor cells. As shown in
Figure 20, the
addition of soluble dlk (0.1 ug/mL) results in an approximately 2-fold
increase in the number
of CSAs initiated by purified fetal liver or BM stem cells over a 2 week time
period (P=
0.001 for dlk vs control and P= 0.01 for dlk vs no additive, Student's t-
test). There was no


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difference in the numbers of CSA observed in BFC012/stem cell cultures with or
without
control fusion protein (mean of no additive/control = 0.96+0.11)
In order to assess the activity of the normal transmembrane form of dlk, a
full-length
dlk cDNA was transfected into BFC012 cells. Expression of the introduced dlk
was
demonstrated at the RNA (Northern blot) and protein levels using both Western
blot and
flow cytometric analyses with rabbit anti-dlk antibodies. The flow cytometry
data are
presented in Figure 21.
Initially, dlk-expressing transfected populations (BFC-dlk) were compared to a
negative control "mock" transfected population of BFC012 cells. A 4- to 6-fold
increase in
to the number of CSAs was observed in two separate experiments. The
maintenance of CSAs
was transient, lasting less than 2 weeks. No further hematopoietic activity
was observed
during an additional 2 weeks of culture. Once dlk-expressing clones had been
identified
from the transfected populations, they were studied for their ability to
support CSAs in
experiments designed to more precisely identify the time course of
hematopoietic activity.
Ficoll-separated, lineage depleted BM was used in these experiments. Five
different
negative control, non-dlk expressing BFC012 cell groups (parental BFC012
cells, two
"mock" transfected populations, and two "mock" transfected clones) and 3 dlk-
expressing
BFC012 cell groups (one transfected population and two clones) were studied.
The data are
presented in Figure 22A. Neither the negative control BFC populations nor the
"mock"
2o transfected BFC clones supported high numbers of CSAs. In contrast, the BFC-
dlk
populations and the two individual dlk-expressing clones supported
significantly greater
numbers of CSAs at all time points studied (P< 0.001 days 3,4, and 5; P< 0.01
days 6 and 7,
Student's t-test). As observed previously, all the CSAs were transient. This
experiment
also indicated that the dlk-promoted hematopoietic activity peaks early, at 4
days, in this
culture system. Three additional experiments using purified (AA4.1+,
line°~-, Sca-1+, c-kit+)
fetal liver stem cells were performed using two individual clones, BFC-dlk-5
and a "mock"
transfected negative control BFC-Zeo-1. The results are presented in Figure
22B. There was
a dramatic and significant difference in the number of CSAs observed in the
BFC-dlk-S
cultures compared to the control line (P< 0.001, days 4, 6, and 8, Student's t-
test). As
3o before, the effect was transient and the CSA declined in number over 2
weeks. AFT024 was
included as a positive control and, in each of the three experiments, verified
the quality of
the input purified stem cells. In the first week of culture the numbers of
CSAs observed on
AFT024 were similar to the numbers in the BFC-dlk5 cultures.


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In order to address the "primitiveness" of the cells that give rise to the CSA
observed
in the BFC-dlk cocultures, a series of in vitro replating experiments were
performed.
Individual wells were harvested at various time points of coculture and the
cells were plated
into semisolid cytokine-containing media. The numbers and lineage compositions
of the
s colonies were scored after 8-12 days. As shown in Figure 23A, the CSAs
obtained from day
4 BFC-dlk-S cocultures contained numerous progenitors capable of extensive
proliferation
and multilineage differentiation. The total number of progenitors from the dlk-
expressing
cultures at day 4 was significantly expanded compared to the content in the
freshly purified
uncultured stem cell population (P= 0.01, Student's t-test). The number and
lineage
composition of colonies derived from parallel day 4 AFT024 cultures was nearly
identical to
BFC-dlk-5 derived colonies. The content of CSAs replated at day 6 from the BFC-
dlk-5
cocultures was devoid of multilineage colonies, although CFU-granulocyte-
macrophages
were maintained at high levels; the progenitor content in the BFC-dlk-5
cocultures continued
to decrease when next sampled at 10 days. In contrast, few progenitors could
be
demonstrated in the BFC-Zeo-1 cultures (P= 0.001, BFC-dlk-5 vs. BFC-Zeo-1,
Student's t-
test) (Figure 23A). Taken together, the data strongly suggest that dlk acts to
promote
stromal-dependent colony-formation by primitive cells capable of yielding
large numbers of
committed progenitors, including those endowed with a high proliferative
capacity and
multilineage differentiation potential. The lack of CSAs and significant
progenitor
2o maintenance in the BFC-Zeo-1 cultures argues that expression of dlk in the
transfected
BFC012 cells is responsible for both their ability to support CSAs and to
generate/maintain
primitive progenitors.
In order to determine if the CSA-containing cultures supported by BFC-dlk-5
contained stem cells capable of in vivo engraftment, portions of the same day
4 cocultures
that were plated into progenitor assays also were used to transplant mice in
competitive
repopulation assays. Shown in Figure 23B are the results from two independent
experiments
analyzed at 10 weeks after transplant. The same BFC-dlk-5 cultures that
contain CSAs and
primitive CFU-high proliferativ potential (HPP)-Mix progenitors also contain
repopulating
stem cells at levels equal to those maintained in parallel AFT024 cocultures.
In addition, a
3o significant difference exists in the levels of repopulating stem cells
derived from dlk-
expressing cocultures compared to non-dlk expressing BFC012 cells (P= 0.05,
Student's t-
test). Mufti-color flow cytometric analyses also demonstrated that both
myeloid and
lymphoid Ly5.2 cells are present in these animals. A subsequent analysis of
these animals at


CA 02340465 2001-02-21
WO 00/11168 go PCT/US99/19052
22 weeks demonstrated lower levels of repopulation with Ly5.2 cells derived
from the
AFT024 and BFC-dlk-5 supported cultures (data not shown). Most significantly,
no
repopulation was observed at any time point in mice that received cells
cocultured on the
non-dlk-expressing monolayers (Figure 23B).
Discussion
As part of an ongoing effort to understand the biology of the hematopoietic
microenvironment, a panel of stromal cell lines from midgestation fetal liver
was established
and characterized. Among these cell lines, the AFT024 line has the ability to
maintain
nearly quantitative levels of transplantable stem cell activity for extended
in vitro time
to periods (Moore, K. A., Ema, H. & Lemischka, I. R.,1997). Because these
cultures are
initiated with highly purified stem cell populations it is likely that the
AFT024-derived
molecular mechanisms responsible for this ability act directly on the stem
cell population.
Other stromal lines that fail to maintain stem cell activity were also
identified. These
observations facilitated a subtractive hybridization approach aimed at
identifying potential
t5 candidate molecules whose collective actions may be responsible for the
AFT024 stem cell
maintenance activity. This effort has identified dlk, a transmembrane molecule
containing
six EGF-like repeat motifs. Although lacking the DSL motif indicative of the
notch ligands
delta and serrate (Tax, F. E., Yeargers, J. J. & Thomas, J. H., 1994), dlk is
most closely
homologous to delta/notch/serrate when compared to other EGF-like repeat
containing
2o molecules (Laborda, J., et al., 1993; Smas, C. M. & Sul, H. S., 1993). The
predominant role
of these types of molecules in cell growth and differentiation led us to
investigate the
potential role of dlk in hematopoiesis. Constitutive expression of
translocated human notch
(Tan-1) is found in a T-cell leukemia (Ellisen, L. W., 1991). Moreover the
expression of
Tan-1 in primitive human stem cells has been demonstrated (Milner, L. A., et
al., 1994).
25 Nevertheless, a functional role in hematopoiesis for the notch ligands
Jagged (Lindsell, C.
E., 1995) and Delta-like-1 ( Bettenhausen, B., et al., 1995) has not been
described. dlk
expression is highly restricted in a panel of stromal cell lines. Two lines,
AFT024 and 2012,
which maintain repopulating stem cell activity in vitro, express dlk, whereas
two non-
supportive cell lines, 2018 and BFC012, do not. Interestingly, the S 17
stromal cell line
3o which is considered to be a potent stem cell supporter (Wineman, J. P., et
al., 1993) does not
express detectable levels of dlk. The S 17 cell line was derived from adult BM
(Collins, L. S.
& Dorshkind, K., 1987)). The other lines described are all derived from fetal
sources
(AFT024, 2012, and NIH 3T3 cells) or from newborn calvaria (PA6 cells). It is
therefore


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possible that dlk acts in a developmentally regulated fashion. An extensive
analysis of dlk
expression in adult BM stroma is currently underway. Taken together, the data
suggest that,
at least in fetal stromal cell types, there exists a correlation between
hematopoietic
supporting ability and the expression of dlk.
The potential activity of both soluble and transmembrane dlk protein, on
highly purified stem cell populations were directly measured using in vitro
and in vivo
assays. Initial experiments designed to ask if dlk can enhance colony
formation in cytokine-
rich semisolid assay systems were negative. These results may indicate that:
(i) progenitor
cells capable of colony formation in semisolid assays do not respond to dlk,
(ii) the
o collection of cytokines present in the semisolid cultures may "mask" an
effects) of added
dlk or (iii) that the soluble form of dlk requires a stromal monolayer to
mediate its effects.
The first possibility can be addressed more extensively in delta-type assays,
where
stem/progenitor cells are first cultured in suspension in serum-free media
containing various
cytokine combinations, with and without dlk, and then replated into colony
assays (Muench,
J. O., Firpo, M. T. & Moore, M. A., 1993). The second possibility can be
addressed by more
extensive studies using subsets of the cytokines present in our initial
studies. These
experiments are underway. As a first step to address the third possibility, we
added soluble
dlk to preestablished BFC012 monolayers. Using both purified BM and fetal
liver stem cell
populations, a significant increase in CSA colony formation was observed in
the dlk
supplemented cultures (Figure 20). This was a surprising result, given that
dlk is a
transmembrane protein; however, before its cDNA cloning, a soluble form of dlk
was
identified as FA1 or fetal antigen 1 (Jensen, C. H., et al., 1994). A role in
hematopoiesis
was not indicated in these studies, but expression was detected in stroma of
placental villi, in
yolk sac blood islands and in fetal liver (Jensen, C. H., et al., 1994). It is
of interest to
determine if a soluble form is produced by the stromal cell lines that express
dlk. An
additional explanation, for the effects observed with the soluble form added
to stromal/stem
cell cocultures, is that they may be facilitated by the Fc portion of the
fusion protein. It is
possible that Fc receptors expressed by some of the hematopoietic cells in the
cultures are
able to sequester and present the dlk-Fc fusion protein more effectively. This
possibility can
3o be addressed by using a different type of soluble dlk protein. These
studies have been
initiated. Alternatively, the soluble dlk may be sequestered and thus
presented by the
stromal cell extracellular matrix. In order to further address the third
possibility, an intact
transmembrane form of dlk was introduced into the BFC012 stromal cell line.
Initially, dlk


CA 02340465 2001-02-21
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transfected BFC012 cell populations were compared to BFC012 cells transfected
with the
selectable marker alone. In these studies, the dlk expressing monolayers were
more effective
at promoting CSA colonies. As with the soluble dlk experiments, the CSAs
appeared early
and were transient. When cells were replated from the BFC-dlk supported
cocultures onto
an AFT024 monolayer a reiteration of the burst of CSAs was seen that was
maintained for 3
weeks. In addition, replating of these cocultures revealed a high progenitor
content (~l in
10) that included a high percentage (43%) of multipotential colonies including
HPP-Mix. In
the BFC-Zeo control populations neither replatable CSA nor CFU progenitors
were
maintained. Further experiments with individual clones from the transfected
populations
to confirmed and extended the results obtained with the populations,
demonstrating highly
significant differences in the numbers of developing CSAs (Figure 22).
However, in two
experiments we observed that one dlk-transfected BFC012 clone, which expresses
a very
high level of dlk, supported fewer CSAs than non-dlk-expressing control cells.
These
cultures also suggested differentiation phenomena, as indicated by the number
of rapidly
accumulating nonadherent cells. Experiments utilizing this cell line were not
included in our
analyses. It is possible that there may be a threshold level of dlk expression
necessary in
these cultures and when it is surpassed the cells differentiate and die
rapidly in the culture
mileau provided by BFC012 cells. In addition, it is possible that an aberrant
form of the dlk
protein is made by this line. Further studies are necessary to clarify this
issue. Nevertheless,
in Figure 22A, dlk-transfected BFC012 cells (one population and two clones)
show a
significant enhancement of CSA formation compared to controls.
The observed low level maintenance of competitive repopulating stem cells in
short-
term dlk-expressing cocultures is of interest even though the activity
diminished over time.
These studies show that the ectopic expression of a single molecule (dlk) in a
previously
nonsupporting stromal cell line restores or enables hematopoietic support.
This is
demonstrated by maintenance of three different stem/progenitor cell
compartments: (i)
CFU-HPP-Mix, (ii) CSA, and (iii) short-term in vivo repopulating stem cells.
It is also of
interest that both qualitatively and quantitatively similar stem/progenitor
cell compartments
are maintained in short-term AFT024 supported cocultures ( Moore, K. A., Ema,
H. &
Lemischka, I. R., 1997).
Two mechanisms underlying the effects of dlk are considered. First, it may be
that
some level of dlk expression is sufficient to retard potent differentiation
signals provided by
the BFC012 cell line. Second, dlk may provide a proliferative stimulus not
normally


CA 02340465 2001-02-21
WO 00/11168 83 PCT/US99/19052
produced by BFC012. A more direct and perhaps relevant assay will be to
eliminate the
expression of dlk in AFT024 cocultures, thus maintaining other components that
make up
the culture "milieu." Studies to evaluate potential neutralizing antibodies
and various dlk
antisense strategies are underway.
The failure of BFC-dlk-5 supported CSAs to persist for periods longer than 1
to 2
weeks also may suggest the existence of other molecules in AFT024 that
facilitate
hematopoiesis. In this regard, it is interesting that our subtraction screen
has yielded
several other clones with expression patterns very similar to dlk. Eventually,
with the
addition of dlk and other AFT024 specific molecules it may be possible to
reconstruct a
1o supportive phenotype. This should lead towards a further understanding of
the in vivo
hematopoietic microenvironment. In summary, it is proposed that dlk represents
one
molecular component responsible for the hematopoietic supportive activities of
the
AFT024 cell line. As such, dlk may define a novel molecular pathway of stem
cell
regulation by the hematopoietic microenvironment.
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1o This invention may be embodied in other forms or carried out in other ways
without
departing from the spirit or essential characteristics thereof. The present
disclosure is
therefore to be considered as in all respects illustrative and not
restrictive, the scope of the
invention being indicated by the appended Claims, and all changes which come
within the
meaning and range of equivalency are intended to be embraced therein.


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SEQUENCE LISTING
<110> Lemischka, Ihor
Moore, Kateri
<120> GENES THAT REGULATE HEMATOPIETIC BLOOD FORMING STEM
CELLS AND USES THEREOF
<130> 2275-1-005PCT
<190> UNASSIGNED
<191> 1999-08-20
<150> US 09/138,132
<151> 1998-08-21
<160> 96
<170> PatentIn Ver. 2.0
<210> 1
<211> 400
<212> DNA
<213> Mus musculus
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aaagcagaaa aaaaggccaa agacttttgg aatggacgtg aaaacatacc tgagatcgat 180
gatcccacat ctggaatctg ggatgaaatc ggccaagtcc aaagacatac tttctgctga 290
agaagttatg cagtggtctc agtctctgga aaaatccttg ccaaccagac aggtcnaaat 300
gtctttggaa gattctaaag tctgattcag tgaggaaaat attgaattct ggttggcttg 360
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gattcgaaan aacacagcca ttctcttc 198
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<213> Mus musculus
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tacggctgcg agaagacnac agaaggggaa ncagctgcgg tggccgcggg agtctgacaa 60
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gcaacggccc caaggtcnan gagaagcggg agaaaatgaa tcggacactc ttnaangatt 180
ggaanacccg tttgagctac ttctngcaga gnncctctgc tcccgggaag cccnnaactg 240
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Page l of 56


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gcctgtggtg gctccgga ccctcccttc ctgctcatcc tggtgtgtac gS ccggag 60
cacctgaacc agagaaacgc cattcgggca tcttggggtg ccatccgcga agcccggggt 120
ttcagagtgc agacgctctt cctcctggga aaacctagaa gacagcagct tgctgacctg 180
tcctcagagt cagcagcaca cagggatatc ttgcaggcct ccttccagga ttcctaccgc 290
aacctcaccc tcaagaccct cagtggactg aactgggtga acaaatactg tcctatggcc 300
cgctacatcc tcaagacgga tgatgacgtg tatgtcaacg tcccananct ggtgtcagag 360
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<900> 5
cccacgcgtc cgatctcctc cagggccacc aagcacctct gaagagccat gttccaagct 60
gccggagccg cccaggccac cccctctcat gaagccaaag gcagcagtgg cagcagcacg 120
gtacagcggt ctaagtcctt tagcttgcgg gctcaggtga aggagacctg tgcagcctgc 180
cagaagactg tgtacccgat ggagcggctg gtggcagaca agctcatttt ccacaactct 290
tgtttctgtt gcaaacactg ccacaccaaa ctcagcctgg gcagttatgc tgcaatgcac 300
ggtgaatttt actgcagacc tcactttcag cagctgttta agagtaaagg caactacgat 360
gaagggtttg gtcgtaaaca gcacaaggag ctctgggccc acaaggaggt ggactcaggc 420
accaagacgg cctgagaccc ctttaacacc cattccctcc cagcacatgg cctcccgctg 980
ggcagtggaa aggagattaa cccgggggcg cggggtggga gaggatgagg ctccctcaca 540
caggtttcag gcataaggct ctgctccagg attccttact tttcccatgg gaggttggcg 600
ttgggaacca gaattggaat tttcaccata ctgtgtcctt tagtccacct catctcaccc 660
cacggctccc tgggaggccc acaagcccag cttccatact taggtgcttt tctccagcaa 720
ggagtcagca tgccctcctc agggtcccaa gctccctcac tgccacctgg gccttgtgta 780
cccccttgtc tccccatcta cctctgcccc ttagcctggt aatgagccac agagactgga B40
agagggagag tgccatntac tgggcctcat agatgccacc tcgctgaggg gggagggctg 900
gggaagaggc aagacagcct gcagccttca gggtctgggg gtcccttgca ccacaaagct 960
aaagctcttg ctagagcctc agctgacagg gtcggcagta gctatgctcc tccatctgtt 1020
gtgctgttct gttgtgatca accctctttt aaaaacattt aaacagcaaa aaaaaaaaaa 1080
a 1081
<210> 6
<211> 793
<212> DNA
<213> Mus musculus
<400> 6
attggtacgc ctgcacgnac cggtccggaa ttcccgggtc gaccacgcgt ccgcccacgc 60
gtccgatctc ctccaggggg caccaagcac ctctgaagaa catgttccaa gctgccggan 120
gcgcccangc caccccctct cntgaagccc aangcagcan tggcagcagc acggtncagc 180
ggtctaagtc ctttaacttg cgggctcagg tgaanganac ctgtgcagcc tgccngaaaa 290
ctgtgtnccc natggancgg ctggtggcag acaanctcnt tttccacaac tcttgtttct 300
gttgcaaaca ctgccacacc aaactcancc tgggcagtta tgctgcnatg caccgtgaat 360
tttactgcag acctcacttt cagcagctgt ttnagaatna aggcnactac natgaaaggt 420
ttggtcgtta acagcacaan ganctctggg cccncnagga agtngactca ggcccnanan 980
aggctganaa ccctttaaca cccattccct cccnncacat tgnctcccnc tnggcagttg 540
gaaaagaaaa taancccngg gcnccgggtt ggganaagaa aaaggtcccc cccnccggtt 600
ttccggcnta agggtctccc ccnnaatccc ttcttttncc cctgggaagt ttgggtttgg 660
gaacccaaat tggaatttcc ccctnccgtn ttcctttntt ccccnccnct cccccccnng 720
gtccctggga aggccccaan ccn 743
<210> 7
<211> 743
<212> DNA
<213> Mus musculus
<900> 7
gggggagtct tgagtcgcat cgcccgttnc gtaagcttgg atcctctana acgggcgccc 60
tttttttttt gttttgctgt ttaaatgttt ttaaaaagaa ggttgatcac cacagaacng 120
cacnacngat ggaggagcat anctnctgcc gaccctgtca gctgangctc tagcaanaac 180
=ttagctttg tg~tgcaagg gaccccnnac cctgaaagct gcaggctgtc ttgcctcttc 290
Page 2 of 56


CA 02340465 2001-02-21
WO 00/1116$ PCT/US99/19052
cccanccctc c~~cctc~ gaagtggcat ctatgaagcc cagtanatgg ca :.tccct 300
cntccnttct c~?tggctca ttaccangct aangggcnga ngttnatngg ganacaaagg 360
ggtncacnat gcccangtgg cantnaagga cttgggacct gaagaaggca tgctgactcc 420
ttgctggana aaaagcacct aanttggaac tgggcttgtg ggcctcccnc ggaaccntgg 480
ggtgagatna a:,tggactat aggaccntat ggtgaaaatt ccaattctgg ttcccaaccc 540
nacctcccat gggaaaaant taggaatcct gcaaccaaac ccttatgcct gaaaacctgt 600
tttnaaggga ncctcatcct cttcccancc ccgngccccc nggggtttaa agggaaaaag 660
ggtttttttn aaagggggtt ccccnnggcc ccccnntggt tnccctnaat ttcccccccc 720
cctnntttgg gccccnaaaa ccn 743
<210> 8
<211> 179
<212> DNA
<213> Mus musculus
<400> 8
ggattccggt ta~gaatgaa attagaagct gtagatctca tggagccacg gttaatatgt 60
gtagccacag tta~tcgaat tattcaccat ctcttgagga tacattttga tggttgggaa 120
gaagagtatg a~~agtgggt agactgtgag tcccctgacc tctatcctgt aggg 179
<210> 9
<211> 711
<212> DNA
<213> Mus musculus
<900> 9
ggggctcagg gaaggccgat cttccgggtg gagggggaag cggcgtgact ggagtggaaa 60
tttttcccaa cacaacttct cggaggcaac atattggaag ggactcgggg aggccggagt 120
ccaaatggaa gtggctgaaa gaaacttctc qccctgctga ttctgagccc cgcgtcgtgc 180
cgcgcgccct caattacctc atcgacctgt ggtcttgaca gaacattctt cacaatccaa 240
aagaaaaagc agactggttc gggatgtttg acggctatga tagctgcagt gaggacacaa 300
gtagcagctc cagctctgag gagagtgaag aagaagttgc tcctttacct tccaatctcc 360
caatcatcaa gaataatgga caagtctaca catacccaga tggtaaatct ggcatggcta 420
cctgtgagat gtgtgggatg gtcggtgtgc gagatgcttt ttactctaaa acgaaacgtt 480
tctgcagtgt tt=ctgttca agaagttact cgtcaaactc taagaaggca agcattctgg 540
ccngacttcn nc~tacgggt tngcctccca cnaagaaagc caaantcctt ccnaaacnac 600
cnttagttgg taaattnnct gcctatgccc ntntccanct accttgcnna atccnnccag 660
acnaaancng gc~.attctgc catctctgtg gaaagggtcc ctgggggtta c 711
<210> 10
<211> 356
<212> DNA
<213> Mus musculus
<900> 10
tacggccgcg anaagacgac agaagggtnc gggctgcgan aagacgacag aaagggggcc 60
tttctccgct gc~cccggcg cgcccggcag ctcctccccg gccatggcgt tcactttcgc 120
ggccttctgc tatatgctgg cgctgctgct. caccgccgcg ctcatcttct tcgccatctg 180
gcacatcata nc.-..~ttgatg agctgaagac cgactacaag aaaccctata gaccagtgca 240
ataccctgaa cc=cttgtcc ttccagaagt actcatccac gcgttcttct gtgtcatgtt 300
tctctgtgcg ca~aattggc tgaccctggg cctcaatatg ccccttttgg catacc 356
<210> 11
<211> 400
<212> DNA
<213> Mus musculus
<900> 11
acggctgcg aa~agacgac agaaggggag tgctcgccgg tcccagcgtt gccataccat 60
cgagatggca tctcgtagcc ggagacccga acacagcgga ccgccggagc tgttttatga 120
ccagaatgaa gc~cggaaat acgttcgcaa ctcacggatg attgacatcc agaccaanat 180
gactgagcga gc~~tggagc tcctctgttt accaganggt cagccttctt acctgttaga 290
~attggctgc gc=~ctgggc tgagtggaaa ttatatctca gaagaaggac actactgggt 300
gggcattgac atcagccctg ccatgttgga tgccgccttg gacgagatac anaaggggac 360
Page 3 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
~gctgctang cgactgg~ aggctc~~tt caaaccggcc 400
<210> 12
<211> 400
<212> DNA
<213> Mus musculus
<400> 12
acggctgcga gaagacgaca gaagggtacg gctgcgagaa gacgacagaa gggtacggct 60
gcganaagac gacagaaggg tacggctgcg agaagacgac agaaggggga ggaaactgta 120
aaagctatgc tc~ggangca gcgcgganct tgattcacct tcacctgctc tggccacccg 180
ctgacccggg g~~=ccggcc gganagcagt canatatgaa cggacgggtg gattatttag 290
tcacggagga agagatcaac ctgacgagag gaccttcggg gctgggcttc aacatcgtcn 300
gtgggacaga tcaacagtat gtctccaacg acagtggcat ctacntcagc cgtatcaaan 360
aagatggggc t?cngcccan gatgggcggc tccaggaagg 400
<210> 13
<211> 1360
<212> DNA
<213> Mus musculus
<900> 13
agctctagtc cccagagatg tcgccactac tgctgctgct gctgtgcctg ctgctgggga 60
atttggagcc tgaggaggcc aaactgatcc gtgtccctct tcaacgaatc caccttggac 120
acagaatctt aaacccactg aatggatggg aacagctggc agagctttct aggacctcca 180
cctctggtgg caacccctcc tttgtgcctc tctccaagtt catgaacacc cagtattttg 290
gaactattgg tttgggaacg cctcctcaga atttcaccgt tgtctttgac acgggttctt 300
ccaacttgtg gg=tccgtcc acgagatgtc atttcttcag tttggcatgc tggtttcacc 360
atcgctttaa tcccaaggcc tccagctcct tcaggcccaa tgggaccaag tttgccattc 920
agtatgggac cgggcggctg agcggaatcc tgagccagga caatctgact atcgggggga 980
tccacgatgc tt~tgtgaca tttggagagg ctctgtggga gcccagcctg atctttgctt 590
tagcccactt tgatgggatc ctgggcctcg gcttccccac tctggctgtg ggcggagttc 600
agcctccgct ggatgcgatg gtggagcaag ggctgctgga gaaacccgtc ttctcctttt 660
acctcaacag ggattctgaa gggtctgatg ggggagagct ggtcctaggg ggctcagacc 720
ccgctcacta cg~acctccc ctcaccttca taccagtcac catccctgcc tactggcagg 780
tccacatgga ga~tgtgaag gtcggcacag ggctgagcct ctgtgcccag ggctgcagtg 890
ccatcctaga cacaggcaca tccctcatca caggacctag tgaggagatc cgggccttga 900
ataaagccat tg~gggatat cccttcctga atgggcagta cttcattcag tgttccaaga 960
cgccaacgct tccccctgtc tccttccacc ttggtggagt ctggtttaac ctcacaggcc 1020
aggactatgt ca~caagatt cttcagagcg atgttggcct ctgcctgttg ggcttccaag 1080
ccttggatat ccccaagcct gcgggacccc tctggatcct tggggacgtc tttttggggc 1140
cctatgtggc tg=ctttgac cgtggggaca aaaacgtcgg cccgcgcgtg ggactggcgc 1200
gtgctcagtc tc~ttcaaca gaccgggcag aaagaaggac tacgcaggcg cagttcttca 1260
aaagacgccc t;~~tagggt acaagctcac cgggccacag cagctatgct tctttccaat 1320
taaacaaact aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1360
<210> 14
<211> 1712
<212> DNA
<213> Mus musculus
<900> 19
tcttcgaaag cc;ggctgag gggaatcctg gacaggggaa tcctggacgt ggagatcgtg 60
agtcatggct gc=_cccgag acgctgatga gatccacaag gacgttcaga actactatgg 120
gaatgtactg aa~acatctg cagacctcca gactaatgct tgtgtcacgc gagccaagcc 180
ggtccccagc ta~atccggg aaagtctgca gaatgtacac gaagacgtta gttcgaggta 240
ttatggctgt gc~~tgactg ttcctgagcg gctggaaaac tgccgaattt tggatctggg 300
tagtgggagt gg~agggatt gctatgtgct tagccagctg gttggtgaga agggacatgt 360
caccggaata ga~atgactg aggtccaggt cgaagtggct aaaacctatc ttgaacacca 420
catggaaaaa tt=3gtttcc aggcacccaa tgtgactttt ctccacggcc gcatcgagaa 480
gttggcagag gc~gggatcc agagtgagag ctatggtatt gtcatatcca actgtgttat 540
caaccttgtt cc=?ataaac aacaagtcct ccaggaggtc tatcgagtgc cgaagcacgg 600
cggggagctc t~=~tcagtg acgtctatgc cagccttgaa gtgccagaag acatcaagtc 660
gcacaaagtt t~~=gggggg aatgcctggg aggcgctctg tactggaagg atcttgccat 720
Page 4 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
cattgcccaa aagattgc tctgccctcc acgtttggtc actgccgata tc ~actgt 780
tgaaaacaag gagctcgaag gggttcttgg tcactgtcgc tttgtgtctg ccacatttcg 840
cctcttcaaa ctccctaaga cagagccagc cgaaagatgc cgagttgttt acaatggagg 900
aatcaaggga catgaaaagg aactaatttt cgatgcaaat ttcacattca aggaaggcga 960
agctgttgca gtggatgagg agacggcagc tgtcctgaag aactcacgtt ttgctccgga 1020
ttttctcttc acacctgttg acgcctcgct gccagctccc caggggccgt tctgagttag 1080
agacaaaggt tctaatcaga gatccattca agcttgcaga ggactctgac aagatgaagc 1190
ccagacatgc acctgaaggc acgggaggct gctgtggcaa gaggaaaaac tgctagatct 1200
acagccagcg cggagcccac cgggctcaag agggtggcta aaggacagtc acagaggctt 1260
cttagcctgc tcttcgccag tgcacagatt atgtgaaggt ggcaaagcca ccacaagcta 1320
gaccactgct aagaataaga gtgactttta gaggatgtta attgaaggtt cacagcaaat 1380
cgcctgcttt tctatttctc tatctcagag ttctggtgcc acctagtggt cagaagtaga 1440
acttggaagc ccaaggttta ctcaaagggc caaaggcatc atcaacgttg tgagaattat 1500
cttccttctg gcctaccaca ggacacctct gggttcttct ctgtggttac caggaagcac 1560
agtacttact aaatttatgc taaccatgac aaaagattgt caactcaaat ttgctaggag 1620
tattctttag gttgctgtct gcaatttttt t=tctgtaac tgaatgaaaa agaaaacaaa 1680
taaaaaataa atttgacttc gaaaaaaaaa as 1712
<210> 15
<211> 1539
<212> DNA
<213> Mus musculus
<400> 15
cggacgcgtg ggggagtctg tggagccccc ggtcaaagac ggcatcctct accagcagca 60
cgtaaagttt ggcaagaaat gctggcgcaa agtgtgggct ctgctgtatg cgggaggccc 120
atcaggggta gctcggctag aaagctggga cgtgcgtgat ggtggcctgg gaccagcagg 180
cgacaggtcc acagggccca gccgtcgagg ggaacgccgg gtcatacgct tggctgactg 290
tgtatctgtc ctgcctgcgg atggcgagag ctgtcccagg gacactggtg ccttcctgat 300
taccaccact gagcgaagcc acctgttggc tgcacagcac cgccagtcct gggtggaccc 360
catctgtcag ctggccttcc cgggtaccgg agaatgttcg tcaggatcag gacaggctga 920
gaatccaaaa aggggctttg ttcccatgga agaaaactct atctactcct cctggcagga 480
agtgaccgaa tttccggtga tcgtgcagaa gacagaagcc acctcccgct gccagctgaa 540
aggaccctac ctcctggtgc tgggccaaga tgacatccaa ctgagggaga catc~aagcc 600
ccaggcctgt tttagctggc cctaccgttt cctgcgcaag tacggctctg acaagggtgt 660
gttctcgttt gaggctggcc gccgctgtga ctcaggtgag ggcctttttg ccttcagtag 720
cccgcgtgcc ccagacatat gtggggttgt ggctgccgcc attgcccgcc agcgggagcg 780
tcttccagag ctggccatgt ccccaccctg ccccctgcct cgggccctct ccct~ccctc 890
cctagagccc cctggagagc ttcgggaggt g~ccccagga tttgagctgc ccactcccag 900
aaagctgcct ctaactgatc ccgggcctca aagcctacca ttgctgctca gccccaacac 960
aagaaggacc ggcatccggt ctctatgcgt ccgtgtgcaa gcagaccagc aagcacacag 1020
gcacggcgga gcatttctat gagaacgtgt gcatgctgga ggccagcctt gggctgacca 1080
atgggggtcc tgaagcccaa gagggccccc ttggtggccg cagccccctt gggcagcctt 1140
atctaccata acactgagga tctgagttgg ccgggctcgg cccaggacag caatctggaa 1200
gcccagtacc ggaggctgct ggaactggag ctggatgagg ccggaagcgc cggccgctct 1260
ggagcgcagg caggcatcaa ggccaagctg gtgaccctgc tgacccgtga acggaagaag 1320
ggccccgccc cctgtgaccg gccctgaagg cctgagcggc cagccactgc aggacagagg 1380
tgatcaccca agaccaggaa caacttcgaa cataacccgt ctactctgac ctgcagggac 1490
aagccaggtg gcccggggag gagccacact ctgccctacc tcctccctca gactgtacag 1500
attgaacagt aataaagctt gcctatcaac ttcaaaaaa 1539
<210> 16
<211> 3599
<212> DNA
<213> Mus abbotti
<400> 16
agagacagcg tgatcccggc ctcccacggg gcagctttta ctgtctaggg aagaaatccc 60
caaagtccat ggagtctgaa gactctgtca agcctcgcta ggaaacctag gagttttaga 120
gggcacttgg caccggaagc tagccgggta g~cggagcct cacctggatt gagt=cacag 180
ctgcctagac aggctcagac taggtgctgg gcacctggga ggaggaggag acattagcag 290
caaaggctgt taacagaagt gcctgcctag ~cttggaggc aagacgctgc tgttcacagt 300
gcgagacgga ggtaggagta taatggctgt ccaggtgctg cggcagatgg tctacttcct 360
actgagtctg ttttctctgg tgcaaggtgc acacagtggc agcccccgag aagacttccg 420
Page 5 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
cttctgtggc cagcgga~ agacccaaca gagcaccctc cactatgatc as .:tcaga 480
gcctcacatc tttgtgtgga acacagagga gaccctcaca attcgtgccc ccttcctggc 590
agccccagat at~ccccgct tcttcccaga gcctagaggg ctctatcact tctgcctcta 600
ctggagtcgc cacactggga gactccactt gcgctatggc aagcatgact acctgcttag 660
tagccaagcc tccagactcc tctgcttcca gaaacaggaq cagagcctga agcagggagc 720
cccgctgatc gccacctctg tcagctcctg gcagattccc cagaacacca gcctgcctgg 780
ggctccgagc ttcatcttct ccttccacaa tgccccacac aaggtctccc acaatgcatc 890
tgtggacatg tgtgatctca agaaggaatt gcagcagctt agcaggtacc tgcagcaccc 900
tcaaaaggct gccaagcggc ccaccgcagc gttcatcagc cagcagttac agagcctgga 960
gtcaaagctg acctctgtga gcttcctggg agacacatta tcctttgagg aggaccgggt 1020
caatgctaca gtgtggaagc tgccacccac agccggtcta gaggatctgc atatccactc 1080
ccagaaggag gaggagcaga gtgaggtcca ggcatactcg ctgttgcttc cccgggccgt 1190
attccagcag accagaggcc gtcgccggga tgacgccaag aggctcctgg tagtagactt 1200
cagcagccaa gctttgttcc aggacaagaa ttctagccaa gtcctgggtg agaaggtctt 1260
gggtattgtc gtgcagaaca ccaaagtcac caacctctca gatccggtgg tactcacctt 1320
ccagcaccag cc~cagccaa aaaatgtgae tctgcagtgc gtgttctggg ttgaagaccc 1380
ggcatcaagc aacacaggga gctggagcag tgcaggctgc gagacagtga gcagagacac 1940
acagacatcc tgcctgtgca accacctgac ctactttgca gtgctgatgg tgtcatccac 1500
agaggtagaa gccactcaca aacactacct cacgctcctg tcctacgtgg gctgtgtcat 1560
ctctgctctg gcttgtgtct tcactatcgc tgcctacctc tgctccagga ggaagtcacg 1620
tgactacacc atcaaagtcc acatgaacct gctgtccgct gtcttcctgc tggacgtgag 1680
cttcctgctc agcgagcctg tggcactgac gggctccgaa gcagcctgtc gcaccagtgc 1790
catgttcctg cacttctccc tgcttgcctg cctctcctgg atgggcctcg agggctacaa 1800
tctctaccga ctggtggtgg aggtcttcgg tacctatgtg cccggctatc tgctcaagct 1860
gagcatcgtg ggctggggtt ttcctgtctt cctggtcact ctggtggcgt tggtggatgt 1920
gaataactac ggccccatta tcctagctgt gcgccggact ccggaacgtg tcacctaccc 1980
ctctatgtgc tggatccggg actccctggt gagctatgtc accaacctgg gcctcttcag 2090
tctggtgttc ctgttcaacc tggctatgct ggccaccatg gtggtgcaga tcctgcggct 2100
tcgcccgcac agccagaact ggccccacgt gctgaccctg ctgggcctca gcctggtcct 2160
tggcctcccc tgggccttgg tcttcttttc ctttgcttcc ggcaccttcc agcttgtcat 2220
cctctacctc ttcagcatca taacttccta ccaaggcttc ctcatcttcc tgtggtactg 2280
gtccatgcgg ttccaggccc aaggcggccc ctcccctctg aagaacaact cagacagcgc 2390
caaactcccc atcagctccg gcagcacctc ctccagccgc atctaagcca ccgccacacc 2400
tcccctccgg gaggacacat gcatggcgtc cgctcacgat gtctgtggcc cagtgctgtg 2960
cccacccagc ct~tgttggt tagtggcata ctagagaagg ccctggtcct tgaaggcgta 2520
gggctgttgc tctggtaggt agatacctag cttgccttgg ggacgactct ggtcctcaaa 2580
gggctcagaa gcacactgcc attctgtttg tggggccgtt tcagtctgga gctaaggcct 2690
tgtctttctg gccacctctg ggtccagctg ttgctgctgg gtgttgagac ctgcagaccc 2700
aagctggggt ta?atctcga aggaggctga cacatccggc ctgagacaca gctaactgtc 2760
ttgacttgct gctctgtctc tgtggtcacc atgcagatcc cgagggtggc actgggggta 2820
aatgttctgg gagaaggttt ggaggcagag caccttagga gctgagcatc tcccccagcc 2880
tttctgcaaa ccctcctctt cattccccat ccccaacccc tcctctgtgt tcccctaacc 2940
ctccacctga agcctggggt cctagaccaa tgctgtgatt tggggtggta gttcccagca 3000
gtttcctggt gccagctatc aacttctgtc tgttgtgtgg gctttggcct ctgactcagg 3060
gcaggtttct gtctgagccc tctctccaag ctgcctcacc tttgctcgca cctcagaggg 3120
acctccatct c~cctgaagc ctcctccctc tggcaagtac tgggatacag ccaccctttc 3180
aacccagcac tc~gaagacc aagacagccc cctctggtga cactggccaa gcttgatctt 3240
tttcctaaga agtggtcttc agatccccgc aggtcgctca gaagacactg ggctgcctag 3300
tgtgaattct gtcctactaa cgtacagtga gcagctcctc acccccaccc ccgcaaaagc 3360
tctcaccaag tc~tggagtg tcaggcaggg ggctggaaat ccaggaggac ttcctgcaaa 3420
aggcagcatt tcatcttgac ctcagccttc aggttgggga gaatgttctt tttaaatacc 3480
agttcatttg tc~~ttgata ttaaagctct ttatagagag tctggaaact gtaggcgatt 3540
gtcgagaaga gaaataaaaa tgagctgtta tctaatgcca tggcaaagca gcacaaaaa 3599
<210> 17
<211> 399
<212> DNA
<213> Mus musculus
<400> 17
agcgacatgg ccccgcccgc gctccaggcc cagcctccag gcggctctca actgaggttc 60
c~gctgttcc tgctgctgtt gctgctgctg ctgtcatggc catcgcaggg ggacgccctg 120
~caatgcctg aa~agcgacc ctccggccct gagtcccaac tcaacgccga cgagctacgg 180
?gtcgcttcc agyacctgct gagccggctg catgccannc atagcgagag gactctaact 290
Page 6 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
ctgaaccaag tcctgac: ctgtcnggat actcantcca cangtgagat tc 3tccac 300
ggccagctgc t..ctccncgt caaccgggcg tcgctgantc anggtctccc cgaacctacc 360
ncgtgcancn agcgctgctc ctgctgacnn cnaangccg 399
<210> 18
<211> 900
<212> DNA
<213> Mus mus::ulus
<900> 18
cggacgcgtg gggaaagcgg gtggggatat acgtgtgctg gtgccctaca attcgacagg 60
cgtcttggga gg;tcgacca ccttgcactg tactctgact tctaatgaga atgtgactat 120
cactcaaata ac:-tggatga agaangattc aggtggaccc cacgctcttg tggctgtctt 180
ccaccccaag a~gyggccca acatcaaaga gccagagagg gtgaaattct tggctgccca 240
acaggatctg aggaacgcat ctctggccat ctcgaactta agtgtnnaag acgaangcat 300
ctatgaatgt ca~attgcca cattccccag aagcagtana agcaccaatg cctggctgaa 360
ngtgcaagcc c~~ctnana acactgcana agccctggac 400
<210> 19
<211> 2313
<212> DNA
<213> Mus musculus
<400> 19
cgaattcctg ggtcgaccca cgcgcccgag agggtagaca gaaaggcggg aaagggctgt 60
gaggtcaaat ggaccccatg gaactgaaca acgtcagcat cgaacccgac ggagacagct 120
gcagcgggga cagtattcag gacagctaca ccggcatgga aaactccgac aaggacgcca 180
tgaacagcca atttgctaat gaagatgccg aaagtcagaa gttcctgaca aatgggtttt 290
tagggaagaa gaagctagcc gattacgcgg atgagcatca ccctggaatg acttcctttg 300
gaatgtcctc atttaacctg agcaacgcca tcatgggcag tgggatctta ggcttgtcct 360
atgccatggc caa:.accggg atcatccttt ttataatcat gctgcttact gtggcaatac 920
tctcgctcta ctcggttcac cttttgctga agacagccaa ggaaggaggg tctctaatct 980
atgaaaaatt gggcgagaaa gcatttggat ggcctgggaa aattggagcc ttcatctcta 590
ttacaatgca gaacattgga gccatgtcaa gctacctctt catcattaag tacgaactgc 600
ctgaagtaat cagagcattc atgggacttg aagaaaacac tggggaatgg tacctcaacg 660
gcaactacct cgtcttattt gtgtccgtgg ggatcatcct cccgctctct ctccttaaaa 720
atttaggcta ccttggctac accagtggat tttctctctc ctgcatggtg tttttcgtca 780
gtgtggtgat tt~~aaaaaa ttccaaattc cctgccctct gcctgctctg gatcacaaca 890
acggaaatct gacgttcaac aacacacttc cgattcacat gatctcgctg cctaatgact 900
cggagagctc gggtgtgaac ttcatgatgg attacgctca ccacaaccca gctgggctgg 960
atgagaagca ggtcgcaggc cctcttcaca gcaatggcgt ggagtacgaa gcccagggtg 1020
ctgagaaatg ccaaccaaaa tactttgtgt tcaattcccg gacggcctat gcaatcccaa 1080
tcctggcttt tgcttttgtc tgccaccctg aggtccttcc catctacagc gagcttaaag 1190
atcgatcccg cagaaagatg cagacggtgt ccaacatttc catctcaggc atgctcgtca 1200
tgtaccttct tgcggccctc tttggttatc tgagcttcta cggggacgtt gaagacgagc 1260
tgctgcatgc tt~~agcaag gtctacacat ttgatacggc tcttctcatg gtgcgcctgg 1320
cagtcctggt ggcagtgaca ctgaccgtgc ccatcgtgct gttcccgatc cgtacttcgg 1380
tgatcacact gc~~tttcca aggaaaccct tcagctggct gaagcatttc gggatcgctg 1990
caatcatcat cgcactcaac aacatcctgg tcatcctcgt gcctaccatc aaatacatct 1500
ttggattcat aggggcttct tctgccacta tgctgatttt cattcttccg gctgcgtttt 1560
atctcaagct cgtcaagaaa gaacctctaa gatcacccca gaagattggg gctttggtct 1620
tccttgtgac tg?aattatt ttcatgatgg gaagcatggc gctcattata ctcgactgga 1680
tctacaaccc gccgaatccc aatcaccact aatcccgggg agacgcgtct ccactggaaa 1790
cagctgaaat tc~~tgaagg acattttagt tgtcttgatt gggatgttag tctgaggaat 1800
tagcaagatt ccaaagacgt ttttctagct ctatcagcac acattttaac ccaggccgtg 1860
cagtgcagtg tgtgatgccc gagttgtgtt tgcagcagct gtgcaagctg aagcctgttg 1920
gctgcgtgtg tt~gtcagca gacaatagcc tgtcccccca tggtcactcc acttctctcc 1980
acccccagat taacaggtaa ttctactctc agaacatcag acaaagacct cctggttggg 2090
atacttgtgg aagagaaaat tatgggtttt gttgggaatg gttttgttgg gaatggtgaa 2100
ggatgcatta aaaattctgt gcgaagtatc atcagttacg gccatctctc actctacacc 2160
aacactaagg gtc~gttgac tagctgaggc agggggatat cttgggctgt ccctgtgagg 2220
atcatgacgt atgacggttg ccagtataga gtacttcatt tcaatactca aggaatagtt 2280
tgcccaacct gcttattaca ccgagttagt gaa 2313
Page 7 of 56


CA 02340465 2001-02-21
WO 00/11168 PCTNS99/19052
<210> 20
<211> 3408
<212> DNA
<213> Mus musculus
<900> 20
gcagctaccg gtccggaatt cccgggtcga cccacgcgtc ccacggctgc gagaagatca 60
cagaaggggg cggtgacgca ggaccaggac tcgcgcgtcc agcggagaag caggagaagc 120
cggcgacctt gc~ctctcag cctgatccct gtcttggcgg cctgaacatt cgcagctgga 180
gagatggcgt tcgtgaagag tggatggtta ct~cggcaga gcaccattct gaaacgctgg 290
aagaagaatt ggttcgacct gtggtcagac ggtcacctga tctactacga tgatcagact 300
cggcagagca tagaggataa ggtccacatg cccgtggact gcatcaatat ccgcacgggg 360
catgagtgcc gggacatcca gcctccagat gggaagccca gagactgtct gctgcagatc 420
gtttgccgag acgggaagac catcagtctc tgtgcagaga gcacagacga ttgcctggca 980
tggaagttta cactgcagga ttccagaaca aacacagctt acgttggttc agcaatcctg 540
tctgaagaga ctgcagtggc cgcgtccccg cctccctacg caacctatgc tacaccnacc 600
cctgaggtct ac7gctatgg tccatacagc ggcgcatacc ccgcaggaac tcaagttgtc 660
~atgccgcca acgggcaggc atatgcagtg ccataccagt acccgtatgc aggagtttat 720
ggacaacagc ctgccaacca agtcatcatc cgcgagcggt accgagacaa tgacagtgac 780
ctggctctgg gcatgctcgc cggggcagcc accggcatgg ccctgggctc tctgttctgg 840
gtcttctaga gccttcaaca ttttctgtgc atagcttctg ttagtcctgt gtgcagtaat 900
ttgatttgca gggcatttct gtttgtgaca agtgtctttc ataataattt aaatagttct 960
tttgaaggtg gtaatctaat aattgtgact gacctgcatg gtaccacaaa gaaagcccga 1020
ggtatgctgt gagtgagagc ctgagtcctt ccgggtacta gcttgcacca agtctttctt 1080
agggactttt ggatggcttt atgtaaacac acccagttaa atgggcaatt tccgtccagt 1190
taggtgcagt gttgaattaa gggatggctt tccttgctat gccaatacta atactgctga 1200
tggaggaaga tgtgtgcaag tgtggtgagg agagtcacag cttctttaac tgtggattct 1260
cttctagacc cctgctgcgt gttaccctag gagctgtggg ctggtggctc ctgcaagact 1320
atggtgtgag gaccctgtaa cgtacctctt ggagcactta ggtacccctt gaagctccta 1380
ggtatcacca gcaggattgg ctgctcagga tgcagagggc caccccctcc ctttaaaaat 1990
tacgctccag taatctgccc agttttattt tcttgttatt cttctgtttg cttttcctgg 1500
ggatgattgg cattagtctg gagttaggaa ttgattcgag tgccggtggg tggaggcatg 1560
cagggagctg tccagcgacc tgctctcagt gtttgtttta ggtatattga ttgccagctc 1620
aggctgcaga gagcctatag agactatttt: tctacttgta aagaaagtat ggtgagggga 1680
attgaggaga gccttgtttg aatgttcctg cctcaggcct cctggggccc acactgcgtg 1740
gtcctgggga ggcttttcct cagcagtgga agggaggccc cgtggctgtc cagagtctca 1800
ggttttgagt gagaaatggg attgggtaga gcatctcagg gatttgttct aatccctcat 1860
gttatgggga tccagccgtg ttctcagtcc agacccgctc acctcagaag agcttaaaac 1920
atttctggtc cccaaatgtg tggcactctg agaagctcac aatctggctt tctaacgaaa 1980
atttgtattt ctaaaattag agaatacatg ttccacgcat ttaaaattta tgttctttca 2040
tgttttaaag ctcccaaatc cagctttgtg actggcatat tttagtttca aacagtaccc 2100
cggcacaaag gtgggatggc acagtgaagg ccccccgccc tctactttgc atagtcttgt 2160
ttctccaggg tgctcccagg aagcattcat tctgactttg ctcagcccag tgcatgcgtg 2220
ctgccttgcc gccgtgctgc tgggtagctc tttcttggtc agatcaagtc ttcaacagat 2280
ctccatgtga gacagttgcc aagtagatga ggtggtgccc atagtgcttt ctcgatactc 2390
cttggggacc tgttgacacc tgcccatttc cagctgacat ttgtttttct gtcatctctg 2400
atagatggga tatgtgacaa catggtacgg acgccgttca gtgtcgcttt aataagcatg 2460
atgctgattt tacatcctgt gctgtatgac tgccatttgc tcacagtgtc accattgcta 2520
aagctccgtg ctttacttac aaaacactaa aaccagtggt tagtgtttca cagtgatttt 2580
aattttagag ttagttactg gcattcctaa agccatagag tactgagtca catccctgaa 2640
gtacttttga aacagaattg tctcctactg tcccatgggt gtgccctgcc tgtctcctgg 2700
ccccaatggg gctagctgta ccaggcagcc atagttgagc ctgatcattc ctgtcaccag 2760
tttgacttga ttatataccc agaatggaat acattcttgg gcatctcagt tcctcagccc 2820
tgatcctcat agacgccacc ctttcgatgg cttttgcggc gtcacttgta cctcagtgag 2880
~cctgcgatt cttgagttag aggggacgac ttgtccagca ttgaggaaca tgtctcctcc 2940
actgagactt aaatgatgat gcagggctgg aagaggctgg ctgctgacac tgcatcgtgg 3000
ctgatgtcat tgctctccta gttctttgat ttaagaacct ttcatatgga aggcctgagg 3060
ctccctcaga tc7~cccttg ccaagaaggc ctggcttagg tcattagtgc ccacagtagc 3120
cttctggagt gtagcaagtt cctgcgtttg agacagaatg gttcagattt attttctaca 3180
tctgttgttg accccatgca ccctctcatt ttgccttcca gtctacgtag atgaaagatg 3240
aaaggcagag gatgcagaca gtcttctttg tgattgcttc tgttattctg ttgcatctac 3300
::gagcccgtt ttctccctgt ctgtgcatac agtatgttta taagtgaact tgttaaaata 3360
~taaatgatc ac~aacccta aaaaaaaaaa aaaaaaaaaa aaaaaaaa 3908
Page 8 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
<210> 21
<211> 878
<212> DNA
<213> Mus musculus
<400> 21
aaatctcagc tgtgcagcct tctagaacct catgtctaca aagtgacaaa ctttcctcag 60
gcacaatcca tcatacagtg gatcaatcag tcagagtcag aggaagaact agtcaaaatt 120
acctcatttt ctgaattcat taacacctta aagaaaaccc acaaatacct aatggaagag 180
agtttcaaaa ctgagccccc agaaagagtg gaagaagcaa agagaatggc tacatatgaa 290
gtcaccacag ctctcagctc cttcttgaag tacctcagag aaacacagca gccagacatg 300
cagctgttgc tactctccat tgctactggt gtaggctatc agttggtaaa cagtatcttt 360
cagcatcttc tggggtgtga tgagttaaac ttcctcttgg atcaaacgga aaataacgaa 420
cataaatacc aagaactgaa aaatatttgc aattacagag cccaggcatt cttggtgctc 980
acagccctaa gagccacagt tgaaatcaca gatgtttcta cagaagagaa aggacaacgt 590
ttgacattaa tacaacaaca tatggggtca ctgttgtctg aagaagttgc acatgtcctc 600
acaaaacatg gagaacatca tgactgggaa aggctggaga atcatttgag attactcatt 660
gagggggact ataaagccac cacccattcc ttacaaatgg atgaagtaaa aaaacaattg 720
caaagtttnt gccatgaaaa gaaacagact tataaacaac aaggtaatga aaacagaaca 780
aaagaaatga tagaaaatgg acatttcctg gacttactcc aacgtttagg cctagacaat 890
tactatccaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 87B
<210> 22
<211> 731
<212> DNA
<213> Mus musculus
<900> 22
actagactct ggtagagaaa aaagtgcctc ggagtggctg ctgaacaacc tcatgactca 60
ccagagtgtg gagcttttca naaaaactca cttntaaacg tcaggaaaca gctcattcgt 120
tcntctaact tatttgtgat gcaagtggaa atagatgtnt atacagctct taaaaagtgg 180
atgttccttc agctggtgcc ntcctggaat ggggtctttg aaaacagctt ttgacaaaaa 290
cagatgtctg gttttccaag tggaaagaag actttgaaag gacgactttc cntgaaactg 300
aacagggaaa accagntcgc gcccntgtnc cgacatttaa ggc~acagta cattatcagt 360
gatctggctt ctgcaaggat cattgagcag gattctctgg taccttcaga atggctggcg 920
gcagtgtata aacagcagtg gctggctatg ctacgggctg aacaagacag tgaagtggcg 980
cctcaagaaa tcaataaaga agancttgag ggaaacagca tg~~~tgtgg tcgaaagctt 590
gccaaagatg gtgngtactg ctggcgctgg acacgcttca att.cggctt tgacctcctt 600
gtgncttaca ccaatcgata catcattttc caacgccata cnctgaacca gccatgttat 660
tggatctgtc agcttacacc tcgaaggagc atagcattta aatgcccttg gcttctttga 720
cagtagtggg a 731
<210> 23
<211> 400
<212> DNA
<213> Mus musculus
<900> 23
gacatttaag gctacagtac attatcagtg atctggcttc tgcaaggatc attgagcagg 60
attctctggt accttcagaa tggctggcgg cagtgtataa acaccagtgg ctggctatgc 120
tacgggctga acaagacagt gaagtggggc ctcaagaaat caataaagaa gaacttgagg 180
gaaacagcat gaggtgtggt cgaaagcttg ccaaagatgg tgag:actgc tggcgctgga 240
cacgcttcaa tttcggcttt gacctccttg tgacttacac cap=cgatac atcattttcc 300
aacgccatac nctgaaccag ccatgttatt ggatctgtca gc~=acacct cgaagganca 360
tancatttaa atgcccttgg cttctttgac agtagtggga 900
<210> 24
<211> 2699
<212> DNA
<213> Mus musculus
<900> 29
tgagcgcaac gcaattaatg tgagttagct cattcattag gcac:.ccagg ctttacactt 60
tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taa~aatttc acacaggaaa 120
Page 9 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
cagctatgac catgatt~ ~ ccaagctcta atacgactca ctatagggaa ac, .ggtacg 180
cctgcaggta ccggtccgga attcccgggt cgacccacgc gtccgacggc tgcgagaaga 290
cgacagaagg gggcaccaca gaggacccct tcaacctaag gaaacaccca ggcttcgata 300
ggaccatgct gcagaggtgg cagaaaaggg agatcagcaa ctttgaatac ctcatgtacc 360
tcaacacact ggccggaagg acctacaatg actacatgca gtatcccgtg tttccctggg 420
tcctcgctga ctacacctca gagatgttga acttgacgaa tcccaagact ttccg;gatc 480
tttctaagcc aatgggggct cagaccaagg aaaggaagtt gaagtttacc cagaggttta 590
aagatgttga aaagattgaa ggagacatga ccgtgcagtg ccactactac acccactatt 600
cctcagccat cattgtcgct tcctacttgg tccgaatgcc accattcacg caggccttct 660
gctccttaca gggcggaagc tttgatgtgg ctgatagaat gttccacagt gtaaagagca 720
cgtgggagtc tgcctccaaa gagaacatga gcgatgtcag ggagctgaca cctgaattct 780
tctacctgcc cgagttttta accaactgta atgcagtgga gtttggctgc atgcaggatg 890
gaacgacact gggggatgtg cagcttcctc cctgggctga cggggatccg aggaaattca 900
tcagcttgca cagacaggct ctggaaagtg acttcgtcag cagcaacctc caccactgga 960
tagacctaat tt~tgggtat aagcagcagg ggccggctgc tgtagaggca gtgaacactt 1020
tccaccccta cttctacggt gatagaatag acctgggcag catcactgac ccgctgatca 1080
agagcaccat cctgggcttc atcagcaact ttggacaggt gcccaagcag atcttcacta 1190
aaccccaccc atccagaaac accacaggga aaaacccagg gcctggaaag gatgcttcca 1200
cccctgtagg cc=cccaggc cactcacagt ccttcctcca cagcctgcca gcactgagac 1260
cctctcaggt cacagtcaaa gatatgtacc ttttctctct agggtcggaa tcccccaaag 1320
gggccatcgg ccacatcgtc cctactgaga agtcaatcct ggcagtggag aagaacaagc 1380
tgctgatgcc ccctctctgg aacaggacct tcagctgggg ctttgatgac ttcagttgct 1490
gcctggggag ctacggctct gacaagatcc tgatgacctt tgagaacctg gctgcctggg 1500
gtccctgtct gtgcgctgta tgcccttccc ccacgatgat cgtcacatcc ggggccagcg 1560
cagtggtgtg catctgggag ctgagcctgg tcaaaggtcg cccgagaggt ctgaaactcc 1620
gacaggcctt gtatggacac actcaggcgg tcacatgtct gacagcctct gtcaccttca 1680
gcctcctggt gagcggatcc caggatcgca cttgtatcct gtgggacctg gaccacctct 1740
ctcgtgtggc ctgcctgcct gtccaccggg aaggcatctc agccattgcc atcagtgatg 1800
tctcgggaac cattgtctcc tgtgccggag cccacttgtc cctgtggaat gtcaatggac 1860
agcccctggc cagtattacc acagcctggg gcccagaagg aaccataacg tgctgctgca 1920
tagtagaggg gccagcgtgg gatgcaagcc acgtgatcat cacggggagt aaggacggaa 1980
tggttcggat ttggaagaca gaggacgtga agatgcctgt tcccaggcag gcagtgatgg 2040
aggagccctc cacggagccc ctaagcccca gaggtcacaa gtgggccaag aatcttgccc 2100
tgagccgaga gctggatgtc agtgttgctc tgagtggcaa gcccagcaag gcaagtcctg 2160
ctgtgacagc tc~ggccatc actaggaacc agagcaagct cttggttggc gatgagaagg 2220
gcgaatcttc tgctggtctg ctgatgggta ggagacaagg agctggaggg accgacctga 2280
aagcctggag ccctggggtc ggcagcaaca ggctacaggc acaagatgat gtgtagcctg 2390
ggctgcttaa ccagagcaag ttttgggggg gctccactcc acacagttct caaggagtcc 2400
ctgatggttt gcaccgtgta ccctaaacat gtctgtagtc tatgggactt ctgtaagaag 2960
gatcttggta gacactgatg ctggaaactg acgctgcatg ggaaatttct acgtggctca 2520
cttcaccaag gcttattgca ctgggaaaag aagagggggt gggtattggt tcatggaaac 2580
caccccactg tctttatttt attaaaactc cattttccga aaaaaaaaaa aaaaaaaaaa 2690
aaaaaaaaa 2649
<210> 25
<211> 999
<212> DNA
<213> Mus musculus
<900> 25
~ccattactt ccacctccca tgaggactgc acctgcggca gcgattttat gtgaacttgg 60
attactgtat ggaaatggag gagtgagaaa gtgtgggaat cataaaagaa ggcagaacca 120
aaagacagag ggatcttggg ccatgagtca cagcaaggaa agcctcccac caaacatctg 180
~actggactc ctacaggaac aagaaatgaa ctatcgtgtt cagcccctga gacttgggct 290
~aacatgatt ttcacattgt ctaccctacc taatagagca gagatgtaaa tattattctt 300
attttagagg tg~gatgcct cagctgcaat gggtgagaac tactcctcat ttattctctt 360
~caaggcaat aaaagagaat ggaccaaaga cagtctgtca tcacatctag tcaaaagagc 920
~aatgtcgca gtacaactct tcaaaagaaa aagaaaaaac aagaaaaaag taataaacag 980
stgtgttctg cttgaaaaa 999
<210> 26
<211> 686
<212> DNA
<213> Mus musculus
Page 10 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
<900> 26
cqgggtcgcg c~~gaattaa gtgagtccat tcttgcacac cgagaaaata tgcgacagat 60
gataagaagt t~~~ctgaac cctttgggaa gagacttgct cagtatctct gatggtagan 120
ggagagctca ta~~cgtaga ggacataatg atggtgaaga ttctttgact catacagatg 180
tcagctcttt c~agacaatg gaccaaatgg tgtctaatat gagaaactat atgcagaaat 240
tagaaagaaa c:=cggtcaa ctttcagtgg atccaaatgg acattcattt tgttcttcct 300
cagttatgac tta~tccaaa ataggagatg aaccgccaaa ggtttttcat gcctcaactc 360
aaactcgtcg a~~tccagga agaataaagg aaaccaggaa agcnatgaga gattctgaca 920
gtgggactag aaaaaatggc tattggtcnt catatccatg aaccgagctc ntgttcntta 980
ataaagtccn nr.wanccnag aaagactggg agatgaaaaa aggtccaccc aggaanttca 590
tcccttatga aatgaaaatt gatgccccct gccttttgaa tgaaggaatt ggccaaattn 600
aaggtttttg aaattttcaa accccggaac aacccaatcc tnggaaaacc cctnnaaatg 660
aaaaaatttt t~gggccctt aaaaan 686
<210> z7
<211> 651
<212> DNA
<213> Mus mus~alus
<900> 27
caaaannacn ty;.gtttagc aaataaaaac tgcccagtgg gacaancgtc gccgggaatc 60
gctgtcagta c~~ccgantt canaaatgcc tggctgttgg ganggncaaa gaaatggnnc 120
gcacagacag n: :aaaaggc cggagaagnc gnntgcccnc gaaaccgaag aaccacanga 180
nccctctccc ccttcgcccc cgttccangc gaacctgacn ancaaangaa nggagangan 290
acccagcana nc~ngcaant ctatganctc ctgactggct ccanggaaan catcccgggc 300
ngggcagaaa aaatccctgg gcnncgcaga actgccccaa gccgaccaag aactgcnnnn 360
tgaancagcn ntctnagaac tgnttgtccn nccannagca nacagaagat gaacatcgac 920
atntctgcct tc~~ctgcat ngcngccctg ggctatggnc acnnaaaaac acggcncngg 980
gaaccggaaa ar.ggaanaan gncnnccnga tngntgantg ncgcggagac acgngacttt 590
cnacnanggg gggggnannc gccccccttn nttgttccna ctgggggggg gaacnncccn 600
aaattcgngc c~~~nngggc ccnagggggg ggggggnccn ttttctttct n 651
<210> 28
<211> 683
<212> DNA
<213> Mus musc;:ius
<900> 28
gggtctgttt ggactgcaga agcaaggggg tgatgtagcc catccttccc tttggagatg 60
ctgagggtgt tt~~tcctgc acccacagcc agggggatgc cactcctccc tccggcttga 120
cctgtttctc tgccgctacc tccctccccg tctcattccg ttgtctgtgg atggtcattg 180
cagtttaaga gcagaacaga tcttttactt tggccgcttg aaaagctagt gtacctcctc 240
tcagtgtttt gg~~tccatc tctcntcctc cantaccttg cttcttactg ataattttgc 300
~ggaattcct aac a ttcaa tgacattttt tttaactact atattgattg tcctttaaaa 360
aagaaaagtg ca:atttatc caaaatgtgt atttcttata cgcttttctt tgttatacca 920
tttcctcagc t~~~ctcttt tatatttgta gganaaactc ccatgttatg gaatcccact 980
gtatgattta taaacagaca atatgtngag tgccttttgc aanaaaaagg gtgtgtttga 590
aatcatccgg ar.~~agccan gaaactgtct ccaaaggaaa cnccnacctc tctgttcctt 600
gctgttntgc tga=catccg ccanaaggtg cttccccccg aattttggtt tggttntgtt 660
tnccgaaaaa t~~=ttccgn ttn 683
<210> 29
<211> 624
<212> DNA
<213> Mus muscuius
<900> 29
aatgcacctc c~=gtattcc cactttcgta ntcatttcgg ttctgatctt gtcaaaccca 60
gcctgaccgc t=~_gacgcc gggatggcct cgttactana cttttctttt taaggaantg 120
ctgttttttt t~~anggttt tcaaaacatt ttgaaaagca tttacttttt tgaccacgan 180
ccatganttt tcaaaaaaat ccggggttgt gtgggttttt ggtttttgtt ttaatttttg 240
gttgcgttgc c_~=tttttt taatggggtt ggccccatga aatgggtgcc ccactcactt 300
~tctganatc gaacngactg tgaatccgct ctttgtcnga anctgaacaa nctgtggctt 360
Page I 1 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
ttttccaact c~gtgtg~ . tttctgaatg ttatgtggta agaccccgcg g5 ,tggcan 920
caactgccct ggaaccccan cccctgcntc catctgtngc tgntgcgccc cacantaaaa 480
cattcanacn t~cctgaaaa gttcttgaaa aaanttaatt anattgtccc nnttttactg 590
ggaaaaaatt anccntactc cnnccaattt tnttcttncn antgggctga aagaaatcct 600
tgttccccan ttttcctttg ggcn 624
<210> 30
<211> 691
<212> DNA
<213> Mus abbotti
<400> 30
atcccagacc aggcaacaag gtcctttgag gtctataatg aaagatctgc attctgatga 60
caatgaggag gaatcagatg aagtggagga taacgacaat gactctgaaa tggagaggcc 120
tgtnaataga ggangcagcc gaagtcgcag agttagctta agtgatggca gcgatagtga 180
aagcagttct ccttcttcac ccctacatca cgaacctcca ccacccttac taaaaaccaa 290
caacaaccag a_tcttgaag tgaaaagtcc nataaagcaa agcaaatcag ataagcaaat 300
aaagaatggt gaatgtgaca aggcatacct agatgaactg gtngagcttc ccagaangtt 360
natgacattg agagaaagac acattctgca gcagatcgtg aaccttatng aagaaactgg 920
gacactttca tatcaccaac acaacatttg attttgatct ttgctcgctg gacaaaacca 980
cagtccgtta actacagagt tcctggaaac atctggaaca tcctgaagat ataaccactg 540
gatgcctcna gaactaatgt gtttnnnnnn nnnnngggtt nnnnnntttg ggtgtgaatt 600
~ttgttcccg ttgtttaaat gaaaaccccc cnaatgatgc n 691
<210> 31
<211> 400
<212> DNA
<213> Mus musculus
<900> 31
gcgagccgac ggagnggctc tacggtggat agcgtgttcc ggaacctatc cctcgaatta 60
gccgagtcag gcagagaggg ggcggggngt gcttccgccc ttgctaggag gggctgcatt 120
gcaggggaga cccagcggca gattctgtca cagacgaggg agaaggcgtg aggagacaaa 180
gccgtcacat ccgcgacagc ttccttcagc agcctcctcc tctccagtcc agagccgacc 240
cccgagcccc tgaggcatcc cttccgtctt cggaacaccc tagtcattca ttgctaacag 300
gggatcatga gggaccctgt aagtagccag tacagctcct ttcttttctg gangatgccc 360
atcccacaac t?gatctgtc ngagctggaa ggcctgggcc 900
<210> 32
<211> 900
<212> DNA
<213> Mus musculus
<400> 32
acggctgcga gaagacgaca gaagggaagc taagggctgg tcgctgcgtc tgaacgccgg 60
gttggacgaa ccgctgtgcg cccttggcgg acgtnagcgg aaagaagatg gcggtgcagg 120
tggtgcaagc tgtgcangcg gttcatcttg antctgacgc tttcctantt tgtctcaacc 180
atgctctgag cacagaaaan gangaagtga tgggtctgtg tataggggan ttgaatgatg 290
acataaggag tcactccnaa tttacataca ctggaacgga aatgcgcaca gtccnagaaa 300
agatggatac catcagaatt gttcatatcc attctgtcat catcttgcgg cgttctgaca 360
aganaaanga ccgtgtnnaa atttctccan ancanctgtc 900
<210> 33
<211> 400
<212> DNA
<213> Mus musculus
<900> 33
gatggcggtc t~~acaggag ttaaagttcc tcgtaatttt cgcttgttgg aagaacttga 60
agaaggacaa aaaggagtag gtgatggtac tgttagctgg ggccttgaag atgatgaaga 120
catgacactt acaaggtgga caggcatgat tattgggcca ccaaggacaa actatgaaaa 180
cagaatatat agcctgaaag tagaatgtgg atctaaatac ccagaagctc ctccatcagt 290
tagatttgta acaaaaatta atatgaatgg gatcaataat tccagtggaa tggtggatgc 300
acggagcata c~aatattag caaaatggca aaattcctat agcattaaag tcatacttca 360
Page 12 of 56


CA 02340465 2001-02-21
WO 00/11168 PCTNS99/19052
aganctaaga ctcttat~_ gtccaaagaa GG=atgaanc 400
<210> 34
<211> 900
<212> DNA
<213> Mus musculus
<400> 39
acggctgcga gaagacgaca gaagggggag c_=ctgcgtc ccagggactc cagtacacca 60
ccatggcgga ttctgagcgt ctctcggccc ccygctgctg gttagcctgc accag~ttct 120
cgcgcaccaa aaagggaatt ctcctgtttg c~aanattat actgtgcctg gtgatcttga 180
tttgcttcag tgcatctaca acatcggcct ac~cctccct gtcggtgatt gagatgatct 240
gtgctgctgt cttacttgtc ttctacacgt ctgacctgca ctccaagata tcattcatca 300
actggccttg gactgacttc ttcagatccc t~ataacaac catcctgtac ctgatcacct 360
ccattgttgt ccttgtagaa agaagaagca g ~ccagagt 900
<210> 35
<211> 400
<212> DNA
<213> Mus musculus
<900> 35
tacggctgcg agaagacgac agaaggggct ccgccttctt ccccacctcc ggctgccggc 60
gacaccggtc tttgcagtcc gggaccccgt gc7atcgtta gcccggtgca cgatgccccc 120
gaaaaaggga ggagatggaa ttaaaccgcc tccaattatt ggaagatttg gaacc~cact 180
gaaaattggt atcgttggat tgccaaatgt tgggaaatct accttcttca atgtattaac 290
caatagtcag gcttcancag aaaacttccc attctgcact attgatccta atgagagcag 300
agtgcctgtg ccagatgaga ggttcgactt tc~ttgccag taccataaac cancaagcaa 360
gatcctgctt tcctaaatgt aatggatatt gctggccttg 900
<210> 36
<211> 900
<212> DNA
<213> Mus musculus
<400> 36
acggctgcga gaagacgacn gaaggggtcc ==~ttcctgc gcggcgtttc agtccctctt 60
gagttgggct gtcgtcnant cgcggcgana c~~gcgcacc gcagccatga cagaagctga 120
tgtnaatccg aangcctatc ccctcncana c::~ccacctc accaagaanc tgctggacct 180
tgttcnacag tcttgttnct acaancagct tc:gaaagga ccnatgaacc nccaaaaccc 240
tcaacagaag catctctgan ttcattgtga ~;~ngcanan ctgaaccttg gagat~~cct 300
gcacctccct ctnctgtcga aaaaaanaat g=~cctacct ntttgtncnn ccaacaggct 360
ttangaaagg cctntgggtt tccagccntc a=ccctgtct 900
<210> 37
<211> 399
<212> DNA
<213> Mus musculus
<900> 37
gaaaggcgcc tagataccgc gatacttgcg g7tatcctgg agctcgactc tcctgttctt 60
gccacatttg actgaagaag gacaacgggg ~caaggtttg aggactgaca gattc~anac 120
ccaggcttcc tcngccccca naaagccacc a-:acctcaca tggaaccaaa ggccccatgc 180
ccanccgccg tcccctcaga gganangaaa t»cgtgttc ttgt.~.ggcgt cactg;cagc 290
gtggccgctc tgaagctgcc tctcctgnta t~~annctgt tggacgttcc tgngatgtgg 300
aagcgccgtt ctgacccggt tctccacatt gacctgcgga tgtcggctga cctca~gcta 360
gtggctcccc tcnatgcnna cactctgngg aa~t 399
<210> 38
<211> 315
<212> DNA
<213> Mus musculus
<900> 38
Page 13 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
acggctgcga gaagacg, , gaangggggc aggatggctc ggtggtacag t~ .agatca 60
anangcatac cccactgagc aagctgatga agcctactgt gagaggcagg gcttgtcnat 120
nangcagatt cgattccggt ttgatggaca accaatcaat gaaacagaca ctccacccca 180
nctggagatg gaggatgagg acaccnttga tgtattccag cagcagacag gaggatcanc 240
ctcccgaggg agcgtcccac acccaaccgt tgtcctgacc tgtgctattg agcagtgacc 300
atgctnccac accca 315
<210> 39
<211> 400
<212> DNA
<213> Mus musculus
<900> 39
acggctgcga gaagacgaca gaaggggaga gcatcatggc gctaagcggt cgactggcat 60
tggccgcgct cagactgtgg ggtccgggag gatgtatctg anctgcttcg gaccanancc 120
tgcagtaggg tggtggtcat ggtgggggcc ggcatcanca cacccagtgg catcccggac 180
ttcagatccc caggggagcg gcctctacag caaccttcag cagtatgaca tcccgtaccc 290
tgaagccatc tttgaacttg gctttttctt tcacaacccc aagccctttt tcatgttggc 300
caaggactgt nccctgngca ctacaggcca atgtcactca ctacttcctg aagctcctcc 360
acgacangga ccgcttctgc gctctntaca canaaatc.ga 900
<210> 90
<211> 349
<212> DNA
<213> Mus musculus
<900> 40
ggccatcaan cgtgctgttg gtcatggccg actcctcaaa gatggacccc tctaccgcct 60
caacactaag gcagccagtg ctgaagggaa agagggctgt gagtcgctct cgtgtttacc 120
tcccgtgtca ctgcttccac acganaaaga caagccggtt gctgaaccaa tccccatctg 180
tagcttctgt cttggtacaa aagaacagaa r_cgggagaag cagcccgagg aactcgtctc 240
ctgcgcggac tgcggcaata ncggtcatcc atcgtgttta aagttctccc canagctnac 300
agtgagagtg aagccttacn gtggcttnca ttgantgtaa aacatcact 399
<210> 91
<211> 397
<212> DNA
<213> Mus musculus
<900> 91
cccacgcgtc cgatctcctc cagggccacc aagcacctct gaagagccat gttccaagct 60
gccggagccg cccaggccac cccctctcat gaagccaaag gcagcagtgg cagcagcacg 120
gtacagcggt ctaagtcctt tagcttgcgg gctcaggtga aggagacctg tgcagcctgc 180
cagaagactg tgtacccgat ggagcggctg gtggcagaca agctcatttt ccacaactct 290
tgtttctgtt gcaaacactg ccacaccaaa ctcagcctgg gcagttatgc tgcaatgcac 300
ggtgaatttt actgcagacc tcactttcag cagctgttta agagtaaagg caactacgat 360
gaaggttttc gtaaacagca caaggactct gggccac 397
<210> 92
<211> 158
<212> PRT
<213> Mus musculus
<400> 42
Met Phe Gln Ala Ala Gly Ala Ala Gln Ala Thr Pro Ser His Glu Ala
1 5 10 15
Lys Gly Ser Ser Gly Ser Ser Thr Val Gln Arg Ser Lys Ser Phe Ser
20 25 30
Leu Arg Ala Gln Val Lys Glu Thr Cys Ala Ala Cys Gln Lys Thr Val
35 90 45
~Ir Pro Met Glu Arg Leu 'Jal Ala Asp Lys Leu Ile ?he His Asn Ser
Page 14 of 56


CA 02340465 2001-02-21
WO 00/I I 168 PCT/US99/19052
50 55 60
Cys Phe Cys Cys Lys His Cys His Thr Lys Leu Ser Leu Gly Ser Tyr
65 70 75 80
Ala Ala Met :?is Gly Glu Phe Tyr Cys Arg Pro His Phe Gln Gln Leu
85 90 95
Phe Lys Ser Lys Gly Asn Tyr Asp Glu Gly Fhe Gly Arg Lys Gln His
100 105 110
Lys Glu Leu Trp Ala His Lys Glu Val Asp Ser Gly Thr Lys Thr Ala
115 120 125
Asp Pro Phe Asn Thr His Ser Leu Pro Ala His Gly Leu Pro Leu Gly
130 135 190
Ser Gly Lys G_u Ile Asn Pro Gly Ala Arg Gly Gly Arg Gly
195 150 155
<210> 43
<211> 1360
<212> DNA
<213> Mus musculus
<400> 93
agctctagtc cccagagatg tcgccactac tgctgctgct gctgtgcctg ctgctgggga 60
atttggagcc tgaggaggcc aaactgatcc gtgtccctct tcaacgaatc caccttggac 120
acagaatctt aaacccactg aatggatggg aacagctggc agagctttct aggacctcca 180
cctctggtgg caacccctcc tttgtgcctc tctccaagtt catgaacacc cagtattttg 290
gaactattgg tttgggaacg cctcctcaga atttcaccgt tgtctttgac acgggttctt 300
ccaacttgtg ggttccgtcc acgagatgtc atttcttcag tttggcatgc tggtttcacc 360
atcgctttaa tcccaaggcc tccagctcct tcaggcccaa tgggaccaag tttgccattc 920
agtatgggac cgggcggctg agcggaatcc tgagccagga caatctgact atcgggggga 480
tccacgatgc ttttgtgaca tttggagagg ctctgtggga gcccagcctg atctttgctt 540
tagcccactt tgatgggatc ctgggcctcg gcttccccac tctggctgtg ggcggagttc 600
agcctccgct ggatgcgatg gtggagcaag ggctgctgga gaaacccgtc ttctcctttt 660
acctcaacag ggattctgaa gggtctgatg ggggagagct ggtcctaggg ggctcagacc 720
ccgctcacta cgtacctccc ctcaccttca taccagtcac catccctgcc tactggcagg 780
tccacatgga gagtgtgaag gtcggcacag ggctgagcct ctgtgcccag ggctgcagtg 890
ccatcctaga cacaggcaca tccctcatca caggacctag tgaggagatc cgggccttga 900
ataaagccat tgggggatat cccttcctga atgggcagta cttcattcag tgttccaaga 960
cgccaacgct tccccctgtc tccttccacc ttggtggagt ctggtttaac ctcacaggcc 1020
aggactatgt catcaagatt cttcagagcg atgttggcct ctgcctgttg ggcttccaag 1080
ccttggatat ccccaagcct gcgggacccc tctggatcct tggggacgtc tttttggggc 1140
cctatgtggc tgtctttgac cgtggggaca aaaacgtcgg cccgcgcgtg ggactggcgc 1200
gtgctcagtc tccttcaaca gaccgggcag aaagaaggac tacgcaggcg cagttcttca 1260
aaagacgccc tggttagggt acaagctcac cgggccacag cagctatgct tctttccaat 1320
taaacaaact aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1360
<210> 44
<211> 419
<212> PRT
<213> Mus musculus
<400> 99
:4et Ser Pro Les Leu Leu Leu Leu Leu Cys Leu Leu Leu Gly Asn Leu
1 5 10 15
Glu Pro Glu G~:: Ala Lys Leu Ile Arg Val Pro Leu Gln Arg Ile His
20 25 30
Leu Gly His ::r? Ile Leu Asn Pry Leu Asn Gly Trp Glu Gln Leu Ala
Page 15 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
35 90 45
Glu Leu Ser Arg Thr Ser Thr Ser Gly ply Asn Pro Ser Phe Val Pro
50 55 60
Leu Ser Lys Phe Met Asn Thr Gln Tyr Phe Gly Thr Ile Gly Leu Gly
65 70 75 BO
Thr Pro Pro Gln Asn Phe Thr Val Val Phe Asp Thr Gly Ser Ser Asn
85 90 95
Leu Trp Val Pro Ser Thr Arg Cys His Phe Phe Ser Leu Ala Cys Trp
100 105 110
Phe His His Arg Phe Asn Pro Lys Ala Ser Ser Ser Phe Arg Pro Asn
115 120 125
Gly Thr Lys Phe Ala Ile Gln Tyr Gig :hr Gly Arg Leu Ser Gly Ile
130 135 190
Leu Ser Gln Asp Asn Leu Thr Ile Gly Gly Ile His Asp Ala Phe Val
145 150 155 160
Thr Phe Gly Glu Ala Leu Trp Glu Pro Ser Leu Ile Phe Ala Leu Ala
165 170 175
His Phe Asp Gly Ile Leu Gly Leu Gly Phe Pro Thr Leu Ala Val Gly
180 185 190
Gly Val Gln Pro Pro Leu Asp Ala Met Val Glu Gln Gly Leu Leu Glu
195 200 205
Lys Pro Val Phe Ser Phe Tyr Leu Asn Arg Asp Ser Glu Gly Ser Asp
210 215 220
Gly Gly Glu Leu Val Leu Gly Gly Sir Asp Pro Ala His Tyr Val Pro
225 230 235 290
Pro Leu Thr Phe Ile Pro Val Thr Ile Pro Ala Tyr Trp Gln Val His
295 250 255.
Met Glu Ser Val Lys Val Gly Thr Gll Leu Ser Leu Cys Ala Gln Gly
260 265 270
Cys Ser Ala Ile Leu Asp Thr Gly Thr Ser Leu Ile Thr Gly Pro Ser
275 280 285
Glu Glu Ile Arg Ala Leu Asn Lys Ala Ile Gly Gly Tyr Pro Phe Leu
290 295 300
Asn Gly Gln Tyr Phe Ile Gln Cys Ser Lys Thr Pro Thr Leu Pro Pro
305 310 315 320
Val Ser Phe His Leu Gly Gly Val Trp Phe Asn Leu Thr Gly Gln Asp
325 330 335
Tyr Val Ile Lys Ile Leu Gln Ser Asc Val Gly Leu Cys Leu Leu Gly
390 39~ 350
Phe Gln Ala Leu Asp Ile Pro Lys Prc Ala Gly Pro Leu Trp Ile Leu
355 360 365
Gly Asp Val Phe Leu Gly Pro Tyr VG: Ala Val Phe Asp Arg Gly Asp
370 375 380
Page 16 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Lys Asn Val Gly Pro Arg Val Gly Leu Ala Arg Ala Gln Ser Arg Ser
385 390 395 y00
Thr Asp Arg Ala Glu Arg Arg Thr Thr Gln Ala Gln Phe Phe Lys Arg
405 910 915
Arg Pro Gly
<210> 95
<211> 3338
<212> DNA
<213> Mus musculus
<400> 95
cggtgacgca ggaccaggac tcgcgcgtcc agcggagaag caggagaagc cggcgacctt 60
gcgctctcag cctgatccct gtcttggcgg cctgaacatt cgcagctgga gagatggcgt 120
tcgtgaagag tggatggtta cttcggcaga gcaccattct gaaacgctgg aagaagaatt 180
ggttcgacct gtggtcagac ggtcacctga tctactacga tgatcagact cggcagagca 290
tagaggataa ggtccacatg cccgtggact gcatcaatat ccgcacgggg catgagtgcc 300
gggacatcca gcctccagat gggaagccca gagactgtct gctgcagatc gtttgccgag 360
acgggaagac catcagtctc tgtgcagaga gcacagacga ttgcctggca tggaagttta 920
cactgcagga ttccagaaca aacacagctt acgttggttc agcaatcctg tctgaagaga 980
ctgcagtggc cgcgtccccg cctccctacg caacctatgc tacaccnacc cctgaggtct 590
acggctatgg tccatacagc ggcgcatacc ccgcaggaac tcaagttgtc tatgccgcca 600
acgggcaggc atatgcagtg ccataccagt acccgtatgc aggagtttat ggacaacagc 660
ctgccaacca agtcatcatc cgcgagcggt accgagacaa tgacagtgac ctggctctgg 720
gcatgctcgc cggggcagcc accggcatgg ccctgggctc tctgttctgg gtcttctaga 780
gccttcaaca ttttctgtgc atagcttctg ttagtcctgt gtgcagtaat ttgatttgca 890
gggcatttct gtttgtgaca agtgtctttc ataataattt aaatagttct tttgaaggtg 900
gtaatctaat aattgtgact gacctgcatg gtaccacaaa gaaagcccga ggtatgctgt 960
gagtgagagc ctgagtcctt ccgggtacta gcttgcacca agtctttctt agggactttt 1020
ggatggcttt atgtaaacac acccagttaa atgggcaatt tccgtccagt taggtgcagt 1080
gttgaattaa gggatggctt tccttgctat gccaatacta atactgctga tggaggaaga 1140
tgtgtgcaag tgtggtgagg agagtcacag cttctttaac tgtggattct cttctagacc 1200
cctgctgcgt gttaccctag gagctgtggg ctggtggctc ctgcaagact atggtgtgag 1260
gaccctgtaa cgtacctctt ggagcactta ggtacccctt gaagctccta ggtatcacca 1320
gcaggattgg ctgctcagga tgcagagggc caccccctcc ctttaaaaat tacgctccag 1380
taatctgccc agttttattt tcttgttatt cttctgtttg cttttcctgg ggatgattgg 1940
cattagtctg gagttaggaa ttgattcgag tgccgqtggg tggaggcatg cagggagctg 1500
tccagcgacc tgctctcagt gtttgtttta ggtatattga ttgccagctc aggctgcaga 1560
gagcctatag agactatttt tctacttgta aagaaagtat ggtgagggga attgaggaga 1620
gccttgtttg aatgttcctg cctcaggcct cctggggccc acactgcgtg gtcctgggga 1680
ggcttttcct cagcagtgga agggaggccc cgtggctgtc cagagtctca ggttttgagt 1740
gagaaatggg attgggtaga gcatctcagg gatttgttct aatccctcat gttatgggga 1800
tccagccgtg ttctcagtcc agacccgctc acctcagaag agcttaaaac atttctggtc 1860
cccaaatgtg tggcactctg agaagctcac aatctggctt tctaacgaaa atttgtattt 1920
ctaaaattag agaatacatg ttccacgcat ttaaaattta tgttctttca tgttttaaag 1980
ctcccaaatc cagctttgtg actggcatat tttagtttca aacagtaccc cggcacaaag 2090
gtgggatggc acagtgaagg ccccccgccc tctactttgc atagtcttgt ttctccaggg 2100
tgctcccagg aagcattcat tctgactttg ctcagcccag tgcatgcgtg ctgccttgcc 2160
gccgtgctgc tgggtagctc tttcttggtc agatcaagtc ttcaacagat ctccatgtga 2220
gacagttgcc aagtagatga ggtggtgccc atagtgcttt ctcgatactc cttggggacc 2280
tgttgacacc tgcccatttc cagctgacat ttgtttttct gtcatctctg atagatggga 2390
tatgtgacaa catggtacgg acgccgttca gtgtcgcttt aataagcatg atgctgattt 2900
tacatcctgt gctgtatgac tgccatttgc tcacagtgtc accattgcta aagctccgtg 2460
ctttacttac aaaacactaa aaccagtggt tagtgtttca cagtgatttt aattttagag 2520
ttagttactg gcattcctaa agccatagag tactgagtca catccctgaa gtacttttga 2580
aacagaattg tctcctactg tcccatgggt gtgccctgcc tgtctcctgg ccccaatggg 2640
gctagctgta ccaggcagcc atagttgagc ctgatcattc ctg:caccag tttgacttga 2700
ttatataccc agaatggaat acattcttgg gcatctcagt tcctcagccc tgatcctcat 2760
agacgccacc ctttcgatgg cttttgcggc gtcacttgta cct:.agtgag tcctgcgatt 2820
Page 17 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
cttgagttag aggggac,. ttgtccagca ttgaggaaca tgtctcctcc ac .agactt 2880
aaatgatgat gcagggctgg aagaggctgg ctgctgacac tgcatcgtgg ctgatgtcat 2990
tgctctccta gttctttgat ttaagaacct ttcatatgga aggcctgagg ctccctcaga 3000
tcgtcccttg ccaagaaggc ctggcttagg tcattagtgc ccacagtagc cttctggagt 3060
gtagcaagtt cctgcgtttg agacagaatg gttcagattt attttctaca tctgttgttg 3120
accccatgca ccc=ctcatt ttgccttcca gtctacgtag atgaaagatg aaaggcagag 3180
gatgcagaca gtcttctttg tgattgcttc tgttattctg ttgcatctac cgagcccgtt 3240
ttctccctgt ctgtgcatac agtatgttta taagtgaact tgttaaaata ttaaatgatc 3300
actaacccta aaaaaaaaaa aaaaaaaaaa aaaaaaaa 3338
<210> 96
<211> 221
<212> PRT
<213> Mus musculus
<900> 46
Met Ala Phe Vai Lys Ser Gly Trp Leu Leu Arg Gln Ser Thr Ile Leu
1 5 10 15
Lys Arg Trp Lys Lys Asn Trp Phe Asp Leu Trp Ser Asp Gly His Leu
20 25 30
Ile Tyr Tyr Asp Asp Gln Thr Arg Gln Ser Ile Glu Asp Lys Val His
35 90 95
Met Pro Val Asp Cys Ile Asn Ile Arg Thr Gly His Glu Cys Arg Asp
50 55 60
Ile Gln Pro Pro Asp Gly Lys Pro Arg Asp Cys Leu Leu Gln Ile Val
65 70 75 BO
Cys Arg Asp Gly Lys Thr Ile Ser Leu Cys Ala Glu Ser Thr Asp Asp
85 90 95
Cys Leu Ala Trp Lys Phe Thr Leu Gln Asp Ser Arg Thr Asn Thr Ala
100 105 110
Tyr Val Gly Ser Ala Ile Leu Ser Glu Glu Thr Ala Val Ala Ala Ser
115 120 125
Pro Pro Pro Tyr Ala Thr Tyr Ala Thr Xaa Thr Pro Glu Val Tyr Gly
130 135 190
Tyr Gly Pro Tyr Ser Gly Ala Tyr Pro Ala Gly Thr Gln Val Val Tyr
195 150 155 160
Ala Ala Asn Gly Gln Ala Tyr Ala Val Pro Tyr Gln Tyr Pro Tyr Ala
165 170 175
Gly Val Tyr Gly Gln Gln Pro Ala Asn Gln Val Ile Ile Arg Glu Arg
180 185 190
Tyr Arg Asp Asn Asp Ser Asp Leu Ala Leu Gly Met Leu Ala Gly Ala
195 200 205
Ala Thr Gly Met Ala Leu Gly Ser Leu Phe Trp Val Phe
210 215 220
<210> 47
<211> 2396
<212> DNA
<213> Mus musc;:lus
Page 18 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
<400> 97
caccacagag gaccccttca acctaaggaa acacccag~~ ttcgatagga ccatgctgca 60
gaggtggcag aaaagggaga tcagcaactt tgaatacc-c atgtacctca acacactggc 120
cggaaggacc tacaatgact acatgcagta tcccgtgttt ccctgggtcc tcgctgacta 180
cacctcagag atgttgaact tgacgaatcc caagactt=~ cgggatcttt ctaagccaat 240
gggggctcag accaaggaaa ggaagttgaa gtttacccag aggtttaaag atgttgaaaa 300
gattgaagga gacatgaccg tgcagtgcca ctactacacc cactattcct cagccatcat 360
tgtcgcttcc tacttggtcc gaatgccacc attcacgcag gccttctgct ccttacaggg 420
cggaagcttt gatgtggctg atagaatgtt ccacagtgta aagagcacgt gggagtctgc 980
ctccaaagag aacatgagcg atgtcaggga gctgacacc~ gaattcttct acctgcccga 590
gtttttaacc aactgtaatg cagtggagtt tggctgcat? caggatggaa cgacactggg 600
ggatgtgcag cttcctccct gggctgacgg ggatccgagg aaattcatca gcttgcacag 660
acaggctctg gaaagtgact tcgtcaqcag caacctccac cactggatag acctaatttt 720
tgggtataag cagcaggggc cggctgctgt agaggcagt? aacactttcc acccctactt 780
ctacggtgat agaatagacc tgggcagcat cactgacccy ctgatcaaga gcaccatcct 890
gggcttcatc agcaactttg gacaggtgcc caagcagatc ttcactaaac cccacccatc 900
cagaaacacc acagggaaaa acccagggcc tggaaagg~t gcttccaccc ctgtaggcct 960
cccaggccac tcacagtcct tcctccacag cctgccagc~ ctgagaccct ctcaggtcac 1020
agtcaaagat at~taccttt tctctctagg gtcggaatc~ cccaaagggg ccatcggcca 1080
catcgtccct actgagaagt caatcctggc agtggagaag aacaagctgc tgatgccccc 1190
tctctggaac aggaccttca gctggggctt tgatgact== agttgctgcc tggggagcta 1200
cggctctgac aagatcctga tgacctttga gaacctggct gcctggggtc cctgtctgtg 1260
cgctgtatgc ccttccccca cgatgatcgt cacatccggg gccagcgcag tggtgtgcat 1320
ctgggagctg agcctggtca aaggtcgccc gagaggtct3 aaactccgac aggccttgta 1380
tggacacact caggcggtca catgtctgac agcctctgtc accttcagcc tcctggtgag 1990
cggatcccag gatcgcactt gtatcctgtg ggacctggac cacctctctc gtgtggcctg 1500
cctgcctgtc caccgggaag gcatctcagc cattgccatc agtgatgtct cgggaaccat 1560
tgtctcctgt gccggagccc acttgtccct gtggaatgtc aatggacagc ccctggccag 1620
tattaccaca gcctqgggcc cagaaggaac cataacgtgc tgctgcatag tagaggggcc 1680
agcgtgggat gcaagccacg tgatcatcac ggggagtaag gacggaatgg ttcggatttg 1790
gaagacagag gacgtgaaga tgcctgttcc caggcaggca gtgatggagg agccctccac 1800
ggagccccta agccccagag gtcacaagtg ggccaagaat cttgccctga gccgagagct 1860
ggatgtcagt gttgctctga gtggcaagcc cagcaaggca agtcctgctg tgacagctct 1920
ggccatcact aggaaccaga gcaagctctt ggttggcgat gagaagggcg aatcttctgc 1980
tggtctgctg atgggtagga gacaaggagc tggagggacc gacctgaaag cctggagccc 2090
tggggtcggc agcaacaggc tacaggcaca agatgatgtg tagcctgggc tgcttaacca 2100
gagcaagttt tgggggggct ccactccaca cagttctcaa ggagtccctg atggtttgca 2160
ccgtgtaccc taaacatgtc tgtagtctat gggacttct; taagaaggat cttggtagac 2220
actgatgctg gaaactgacg ctgcatggga aatttctacg tggctcactt caccaaggct 2280
tattgcactg ggaaaagaag agggggtggg tattggttca tggaaaccac cccactgtct 2390
ttattttatt aaaactccat tttccgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2396
<210> 98
<211> 693
<212> PRT
<213> Mus musculus
<900> 48
Thr Thr Glu Asp Pro Phe Asn Leu Arg Lys His Pro Gly Phe Asp Arg
1 5 10 15
Thr Met Leu Gln Arg Trp Gln Lys Arg Glu lle Ser Asn Phe Glu Tyr
20 25 30
Leu Met Tyr Leu Asn Thr Leu Ala Gly Arg Thr Tyr Asn Asp Tyr Met
35 40 95
Gln Tyr Pro Val Phe Pro Trp Val Leu Ala Asp Tyr Thr Ser Glu Met
50 55 60
Leu Asn Leu Thr Asn Pro Lys Thr Phe Arg Asp Leu Ser Lys Pro Met
65 70 75 80
Gly Ala Gln ~!:r Lys Glu Arg Lys Leu Lys Fhe Thr Gln Arg Phe Lys
Page 19 of 56


CA 02340465 2001-02-21
WO 00/i 1168 PCT/US99/19052
B 90
Asp Val Glu Lys Ile Glu Gly Asp Met '"hr Val Gln Cys His Tyr Tyr
100 105 110
Thr His Tyr Ser Ser Ala Ile Ile Val Ala Ser Tyr Leu Val Arg Met
115 120 125
Pro Pro Phe Thr Gln Ala Phe Cys Ser Leu Gln Gly Gly Ser Phe Asp
130 135 190
Val Ala Asp Arg Met Phe His Ser Val Lys Ser Thr Trp Glu Ser Ala
195 150 155 160
Ser Lys Glu Asn Met Sex Asp Val Arg Glu Leu Thr Pro Glu Phe Phe
165 170 175
Tyr Leu Pro Glu Phe Leu Thr Asn Cys Asn Ala Val Glu Phe Gly Cys
180 185 190
Met Gln Asp Gly Thr Thr Leu Gly Aso Val Gln Leu Pro Pro Trp. Ala
195 200 205
Asp Gly Asp Pro Arg Lys Phe Ile Ser Leu His Arg Gln Ala Leu Glu
210 215 220
Ser Asp Phe Val Ser Ser Asn Leu His His Trp Ile Asp Leu Ile Phe
225 230 235 240
Gly Tyr Lys Gln Gln Gly Pro Ala Ala Val Glu Ala Val Asn Thr Phe
245 250 255
His Pro Tyr Phe Tyr Gly Asp Arg Ile Asp Leu Gly Ser Ile Thr Asp
260 265 270
Pro Leu Ile Lys Ser Thr Ile Leu Gly Phe Ile Ser Asn Phe Gly Gln
275 280 285
Val Pro Lys Gln Ile Phe Thr Lys Pro His Pro Ser Arg Asn Thr Thr
290 295 300
Gly Lys Asn Pro Gly Pro Gly Lys Asp Ala Ser Thr Pro Val Gly Leu
305 310 315 320
Pro Gly His Ser Gln Ser Phe Leu His Ser Leu Pro Ala Leu Arg Pro
325 330 335
Ser Gln Val Thr Val Lys Asp Met Tyr Leu Phe Ser Leu Gly Ser Glu
390 395 350
Ser Pro Lys Gly Ala Ile Gly His Ile Val Pro Thr Glu Lys Ser Ile
355 360 365
Leu Ala Val Glu Lys Asn Lys Leu Leu Met Pro Pro Leu Trp Asn Arg
370 375 380
Thr Phe Ser Trp Gly Phe Asp Asp Phe Ser Cys Cys Leu Gly Ser Tyr
385 390 395 400
Gly Ser Asp Lys Ile Leu Met Thr Phe Glu Asn Leu Ala Ala Trp Gly
905 910 415
Pro Cys Leu Cys Ala Val Cys Pro Ser Pro Thr Met lle Val Thr Ser
920 42~ 930
Page 20 of 56


CA 02340465 2001-02-21
WO 00/11168 PCTNS99/19052
Gly Ala Ser Ala Val Val Cys Ile Trp Glu Leu Ser Leu Val Lys ply
935 490 995
Arg Pro Arg Gly Leu Lys Leu Arg Gln Ala Leu Tyr Gly His Thr ~ln
950 955 460
Ala Val Thr Cys Leu Thr Ala Ser Val Thr Phe Ser Leu Leu Val Ser
465 970 475 980
Gly Ser Gln Asp Arg Thr Cys Ile Leu Trp Asp Leu Asp His Leu Ser
485 990 495
Arg Val Ala Cys Leu Pro Val His Arg Glu Gly Ile Ser Ala Ile Ala
500 505 510
Ile Ser Asp Val Ser Gly Thr Ile Val Ser Cys Ala Gly Ala His Leu
515 520 525
Ser Leu Trp Asn Val Asn Gly Gln Pro Leu Ala Ser Ile Thr Thr Ala
530 535 590
Trp Gly Pro Glu Gly Thr Ile Thr Cys Cys Cys Ile Val Glu Gly Pro
595 550 555 560
Ala Trp Asp Ala Ser His Val Ile Ile Thr Gly Ser Lys Asp Gly Met
565 570 575
Val Arg Ile Trp Lys Thr Glu Asp Val Lys Met Pro Val Pro Arg Gln
580 585 590
Ala Val Met Glu Glu Pro Ser Thr Glu Pro Leu Ser Pro Arg Gly His
595 600 605
Lys Trp Ala Lys Asn Leu Ala Leu Ser Arg Glu Leu Asp Val Ser val
610 615 620
Ala Leu Ser Gly Lys Pro Ser Lys Ala Ser Pro Ala Val Thr Ala ~eu
625 630 635 690
Ala Ile Thr Arg Asn Gln Ser Lys Leu Leu Val Gly Asp Glu Lys Gly
645 650 655
Glu Ser Ser Ala Gly Leu Leu Met Gly Arg Arg Gln Gly Ala Gly Gly
660 665 670
Thr Asp Leu Lys Ala Trp Ser Pro Gly Val Gly Ser Asn Arg Leu Gln
675 680 685
Ala Gln Asp Asp Val
690
<210> 49
<211> 2363
<212> DNA
<213> Mus musculus
<900> 49
cagaggcagg caggacaggc actgcasgca ctgagagcct tgcagtcaga agggaagcac 60
asacaggaag aggcagtgag gagaaaagga gcasaactga gacaagcaat ggagatccct 120
gaggagtgct ggccaacggc tgaagtgtcc ctaaaagaca tcactgaaat aatggagaga 180
catctcagtc acatggaacg gaccctgtct cacagtcaaa agctctcaga tggagacctg 290
gtaagatggg catctggagg gctggtcctg cagggaattt ataagaccaa ccacccaaga 300
Page 21 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
agcctgattc agaagag, agagctactc agtgtcccta agcagttctc a~ gctggc 360
ccagtacatg gcacagagat aaaaacaaag gaattttcat cttttcaaga acaagccatg 420
tttacacaga ctatagagag ggtgggtttc agttcaaccc ccttagttaa aggagaaggt 480
tggggactta gtctagaagc tggtatgggt cacaacaaac agacagaatc tgaagataac 590
taccagtccc attcaaagca aacttatttt tgctcagcca ggttcagcta catcccattg 600
gccacctgcc attttcacat caatgatctt gaactctccc aggctgctct ccaggaacta 660
aaaagtattg aagaaatcct ggagcagact acacaccacc gagatggact acccttactg 720
aggcacaggg ctaaaaactt tttccacagg tttggctctc atgctaacca aggccctgtg 780
cacctggggg gaatctactg ctggaaagcc atttcagaag gtttcaaaag tgagcacttg 890
gctgatgtaa agcagcaagc agaagagtct ttgaatattt acattatggg cagttatagt 900
ggctttggag ttaaagttgg tgcgagtgta aatataacaa attcaaaatc aaaaacagca 960
ttttacagta aaactcatct aaactcgcaa accaaggtac aactatctgt agccaagata 1020
ggtggaccag cagaagcaga tggaattgcc cagtggacag ctggccttgt agctagcaat 1080
caaacctggt ctcttattga taggaaactg cagttggtac ctatttggga cattatcctg 1190
tccagtcaca gaactgaatt taagaatgct cttcaactgg ctaactgcct caaagaccac 1200
tacactgctc tga~tgaact agctgcccag attcaagaag gggaagaatt tctgactgct 1260
agaaaagaay ctaagctttt cctaaagaat gtgaaaggct gggaggtttc tgatcctgaa 1320
gaacagctta ggaagttagt agattttatg caaacattga gtcaaaaaat aaaaagttat 1380
gacatttgga ttaacacatg cctcatagat tgggatctgc aaaattttct aataaacatt 1990
gtcaacttct gcaaaaattc acccacttat aaaactcact ttattaaatc tcagttgtgc 1500
agccttctag aacctcatgt ctacaaagtg acaaactttc ctcaggcaca atccatcata 1560
cagtggatca atcagtcaga gtcagaggaa gaactagtca aaattacctc attttctgaa 1620
ttcattaaca ccttaaagaa aacccacaaa tacctaatgg aagagagttt caaaactgag 1680
cccccagaaa gagtggaaga agcaaagaga atggctacat atgaagtcac cacagctctc 1740
agctccttct tgaagtacct cagagaaaca cagcagccag acatgcagct gttgctactc 1800
tccattgcta ctggtgtagg ctatcagttg gtaaacagta tttttcagca tcttctgggg 1860
tgtgatgagt taaacttcct cttggatcaa atggaaaata acgaacataa ataccaagaa 1920
ctgaaaaata tttgcaatta cagagcccag gcattcttgg tgctcacagc cctaagagcc 1980
acagttgaaa tcacagatgt ttctacagaa gagaaaggac aacgtttgac attaatacaa 2090
caacatatgg ggtcactgtt gtctgaagaa gttgcacatg tcctcacaaa acatggagaa 2100
catcatgact gggaaaggct ggagaatgat ttgagattac tcattgaggg ggactataaa 2160
gccaccaccc attccttaca aatggatgaa gtaaaaaaac aattgcaaag tttntgccat 2220
gaaaagaaac agacttataa acaacaaggt aatgaaaaca gaacaaaaga aatgatagaa 2280
aatggacatt tcctggactt actccaacgt ttaggcctag acaattacta tccaaaaaaa 2340
aaaaaaaaaa aaaaaaaaaa aaa 2363
<210> 50
<211> 778
<212> PRT
<213> Mus musculus
<900> 50
Gln Arg Gln Aia Gly Gln Ala Leu Xaa Ala Leu Arg Ala Leu Gln Ser
1 5 10 15
Glu Gly Lys Hi_-- Xaa Gln Glu Glu Ala Val Arg Arg Lys Gly Ala Xaa
20 25 30
Leu Arg Gln Aia Met Glu Ile Pro Glu Glu Cys Trp Pro Thr Ala Glu
35 40 95
Val Ser Leu Lye Asp Ile Thr Glu Ile Met Glu Arg His Leu Ser His
50 55 60
Met Glu Arg T!:r Leu Ser His Ser Gln Lys Leu Ser Asp Gly Asp Leu
65 ?0 75 80
Val Arg Trp Ala Ser Gly Gly Leu Val Leu Gln Gly Ile Tyr Lys Thr
85 90 95
Asn His Pro Arg Ser Leu Ile Gln Lys Arg Glu Glu Leu Leu Ser 'Jal
10:. 105 110
Pro Lys Gln P::e Ser Leu Ala Gly Pro Val His Gly Thr Glu Ile Lys
Page 22 of 56


CA 02340465 2001-02-21
WO 00/11168 PCTNS99/19052
115 120 125
Thr Lys Glu Phe Ser Ser Phe Gln Glu Gln Ala Met Phe Thr Gln Thr
130 135 140
Ile Glu Arg Val Gly Phe Ser Ser Thr Pro Leu Val Lys Gly Glu Gly
145 150 155 160
Trp Gly Leu Ser Leu Glu Ala Gly Met Gly His Asn Lys Gln Thr Glu
165 170 175
Ser Glu Asp Asn Tyr Gln Ser His Ser Lys Gln Thr Tyr Phe Cys Set
180 185 190
Ala Arg Phe Ser Tyr Ile Pro Leu Ala Thr Cys His Phe His Ile Asn
195 200 205
Asp Leu Glu Leu Ser Gln Ala Ala Leu Gln Glu Leu Lys Ser Ile Glu
210 215 220
Glu Ile Leu Glu Gln Thr Thr His His Arg Asp G1y Leu Pro Leu Leu
225 230 235 290
Arg His Arg Aia Lys Asn Phe Phe His Arg Phe Gly Ser His Ala Asn
295 250 255
Gln Gly Pro Val His Leu Gly Gly Ile Tyr Cys Trp Lys Ala Ile Ser
260 265 270
Glu Gly Phe Lys Ser Glu His Leu Ala Asp Val Lys Gln Gln Ala Glu
275 280 285
Glu Ser Leu Asn Ile Tyr Ile Met Gly Ser Tyr Ser Gly Phe Gly Val
290 295 300
Lys Val Gly Ala Ser Val Asn Ile Thr Asn Ser Lys Ser Lys Thr Ala
305 310 315 320
Phe Tyr Ser Lys Thr His Leu Asn Ser Gln Thr Lys Val Gln Leu Ser
325 330 335
Val Ala Lys Ile Gly Gly Pro Ala Glu Ala Asp Gly Ile Ala Gln Trp
390 345 350
Thr Ala Gly Leu Val Ala Ser Asn Gln Thr Trp Ser Val Ile Asp Arg
355 360 365
Lys Leu Gln Leu Val Pro Ile Trp Asp Ile Ile Leu Ser Ser His Arg
370 375 380
Thr Glu Phe Lys Asn Ala Leu Gln Leu Ala Asn Cys Leu Lys Asp His
385 390 395 900
Tyr Thr Ala Leu Thr Glu Leu Ala Ala Gln Ile Gln Glu Gly Glu Glu
405 410 415
Phe Leu Thr Ala Arg Lys Glu Ala Lys Leu Phe Leu Lys Asn Val Lys
920 925 930
Gly Trp Glu Val Ser Asp Pro Glu Glu Gln Leu Arg Lys Leu Val Asp
935 940 495
Phe Met Gln Thr Leu Ser Gln Lys Ile Lys Ser Tyr Asp Ile Trp Ile
950 955 960
Page 23 of 56


CA 02340465 2001-02-21
WO 00/11 lb8 PCTNS99/19052
Asn Thr Cys Leu Ile Asp Trp Asp Le~~ ~ln Asn Phe Leu Ile Asn .le
965 470 475 480
Val Asn Phe Cys Lys Asn Ser Pro Th= :yr Lys Thr His Phe Ile :.ys
485 490 995
Ser Gln Leu Cys Ser Leu Leu Glu Prc His Val Tyr Lys Val Thr Asn
500 50~ 510
Phe Pro Gln Ala Gln Ser Ile Ile G1.~. Trp Ile Asn Gln Ser Glu Ser
515 520 525
Glu Glu Glu Leu Val Lys Ile Thr Ser Phe Ser Glu Phe Ile Asn Thr
530 535 590
Leu Lys Lys Thr His Lys Tyr Leu Mss ~lu Glu Ser Phe Lys Thr Glu
545 550 555 X60
Pro Pro Glu Arg Val Glu Glu Ala Lys Arg Met Ala Thr Tyr Glu Val
565 570 575
Thr Thr Ala Leu Ser Ser Phe Leu Lys Tyr Leu Arg Glu Thr Gln Gln
580 585 590
Pro Asp Met Gln Leu Leu Leu Leu Ser Ile Ala Thr Gly Val Gly Tyr
595 600 605
Gln Leu Val Asn Ser Ile Phe Gln His Leu Leu Gly Cys Asp Glu Leu
610 615 620
Asn Phe Leu Leu Asp Gln Met Glu Asn Asn Glu His Lys Tyr Gln Glu
625 630 635 690
Leu Lys Asn Ile Cys Asn Tyr Arg Ala Gln Ala Phe Leu Val Leu Thr
695 650 655
Ala Leu Arg Ala Thr Val Glu Ile Th= Asp Val Ser Thr Glu Glu Lys
660 665 670
Gly Gln Arg Leu Thr Leu Ile Gln Gln His Met Gly Ser Leu Leu Ser
675 680 685
Glu Glu Val Ala His Val Leu Thr Lys His Gly Glu His His Asp ':'rp
690 695 700
Glu Arg Leu Glu Asn Asp Leu Arg Leu Leu Ile Glu Gly Asp Tyr ~ys
705 710 715 720
Ala Thr Thr His Ser Leu Gln Met Asp Glu Val Lys Lys Gln Leu Gln
725 730 735
Ser Xaa Cys His Glu Lys Lys Gln Th= Tyr Lys Gln Gln Gly Asn Glu
790 745 750
Asn Arg Thr Lys Glu Met Ile Glu Asn Gly His Phe Leu Asp Leu ~eu
755 760 765
Gln Arg Leu Gly Leu Asp Asn Tyr Tyr Pro
770 775
<210> 51
<211> 1712
Page 24 of 56


CA 02340465 2001-02-21
WO 00/1116$ PCT/US99/19052
<z12> DNA
<213> Mus musculus
<900> 51
tcttcgaaag ccgggctgag gggaatcctg gacaggggaa tcctggacgt ggagatcgtg 60
agtcatggct gcttcccgag acgctgatga gatccacaag gacgttcaga actactatgg 120
gaatgtactg aagacatctg cagacctcca gactaatgct tgtgtcacgc gagccaagcc 180
ggtccccagc tacatccggg aaagtctgca gaatgtacac gaagacgtta gttcgaggta 290
ttatggctgt ggtctgactg ttcctgagcg gctggaaaac tgccgaattt tggatctggg 300
tagtgggagt ggcagggatt gctatgtgct tagccagctg gttggtgaga agggacatgt 360
caccggaata gacatgactg aggtccaggt cgaagtggct aaaacctatc ttgaacacca 920
catggaaaaa tttggtttcc aggcacccaa tgtgactttt ctccacggcc gcatcgagaa 980
gttggcagag gctgggatcc agagtgagag ctatggtatt gtcatatcca actgtgttat 590
caaccttgtt cctgataaac aacaagtcct ccaggaggtc tatcgagtgc tgaagcacgg 600
cggggagctc tatttcagtg acgtctatgc cagccttgaa gtgccagaag acatcaagtc 660
gcacaaagtt ttatgggggg aatgcctggg aggcgctctg tactggaagg atcttgccat 720
cattgcccaa aagattgggt tctgccctcc acgtttggtc actgccgata tcattactgt 780
tgaaaacaag gagctcgaag gggttcttgg tgactgtcgc tttgtgtctg ccacatttcg 890
cctcttcaaa ctccctaaga cagagccagc cgaaagatgc cgagttgttt acaatggagg 900
aatcaaggga catgaaaagg aactaatttt cgatgcaaat ttcacattca aggaaggcga 960
agctgttgca gtggatgagg agacggcagc tgtcctgaag aactcacgtt ttgctccgga 1020
ttttctcttc acacctgttg acgcctcgct gccagctccc caggggccgt tctgagttag 1080
agacaaaggt tctaatcaga gatccattca agcttgcaga ggactctgac aagatgaagc 1190
ccagacatgc acctgaaggc acgggaggct gctgtggcaa gaggaaaaac tgctagatct 1200
acagccagcg cggagcccac cgggctcaag agggtggcta aaggacagtc acagaggctt 1260
cttagcctgc tcttcgccag tgcacagatt atgtgaaggt ggcaaagcca ccacaagcta 1320
gaccactgct aagaataaga gtgactttta gaggatgtta attgaaggtt cacagcaaat 1380
cgcctgcttt tctatttctc tatctcagag ttctggtgcc acctagtggt cagaagtaga 1440
acttggaagc ccaaggttta ctcaaagggc caaaggcatc atcaacgttg tgagaattat 1500
cttccttctg gcctaccaca ggacacctct gggttcttct ctgtggttac caggaagcac 1560
agtacttact aaatttatgc taaccatgac aaaagattgt caactcaaat ttgctaggag 1620
tattctttag gttgctgtct gcaatttttt tctctgtaac tgaatgaaaa agaaaacaaa 1680
taaaaaataa atttgacttc gaaaaaaaaa as 1712
<210> 52
<211> 336
<212> PRT
<213> Mus musculus
<900> 52
Met Ala Ala Ser Arg Asp Ala Asp Glu Ile His Lys Asp Val Gln Asn
1 5 10 15
Tyr Tyr Gly Asn Va1 Leu Lys Thr Ser Ala Asp Leu Gln Thr Asn Ala
20 25 30
Cys Val Thr Arg Ala Lys Pro Val Pro Ser Tyr Ile Arg Glu Ser Leu
35 90 45
Gln Asn Val His Glu Asp Val Ser Ser Arg Tyr Tyr Gly Cys Gly Leu
50 55 60
Thr Val Pro Glu Arg Leu Glu Asn Cys Arg Ile Leu Asp Leu Gly Ser
65 70 75 80
Gly Ser Gly Arg Asp Cys Tyr Val Leu Ser Gln Leu Val Gly Glu Lys
85 90 95
Gly His Val Thr Gly Ile Asp Met Thr Glu Val Gln Val Glu Val Ala
100 105 110
Lys Thr Tyr Leu Glu His His Met Glu Lys Phe Gly Phe Gln Ala Pro
115 120 :25
Page 25 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Asn Val Thr Phe Les is Gly Arg Ile Glu Lys Leu Ala Glu 3 Gly
130 135 140
Ile Gln Ser Glu Ser Tyr Gly Ile Val Ile Ser Asn Cys Val Ile Asn
195 150 155 160
Leu Val Pro Asp Lys Gln Gln Val Leu Gln Glu Val Tyr Arg Val Leu
165 170 175
Lys His Gly Gly Glu Leu Tyr Phe Ser Asp Val Tyr Ala Ser Leu Glu
180 185 190
Val Pro Glu Asp Ile Lys Ser His Lys Val Leu Trp Gly Glu Cys Leu
195 200 205
Gly Gly Ala Leu Tyr Trp Lys Asp Leu Ala Ile Ile Ala Gln Lys Ile
210 215 220
Gly Phe Cys Pro Pro Arg Leu Val Thr Ala Asp Ile Ile Thr Val Glu
225 230 235 290
Asn Lys Glu Leu Glu Gly Val Leu Gly Asp Cys Arg Phe Val Ser Ala
295 250 255
Thr Phe Arg Leu Phe Lys Leu Pro Lys Thr Glu Pro Ala Glu Arg Cys
260 265 270
Arg Val Val Tyr Asn Gly Gly Ile Lys Gly His Glu Lys Glu Leu Ile
275 280 285
Phe Asp Ala Asn Phe Thr Phe Lys Glu Gly Glu Ala Val Ala Val Asp
290 295 300
Glu Glu Thr Ala Ala Val Leu Lys Asn Ser Arg Phe Ala Pro Asp Phe
305 310 315 320
Leu Phe Thr Pro Val Asp Ala Ser Leu Pro Ala Pro Gln Gly Pro Phe
325 330 335
<210> 53
<211> 3599
<212> DNA
<213> Mus musculus
<900> 53
agagacagcg tgatcccggc ctcccacggg gcaqctttta ctgtctaggg aagaaatccc 60
caaagtccat ggagtctgaa gactctgtca agcctcgcta ggaaacctag gagttttaga 120
gggcacttgg caccggaagc tagccgggta ggcggagcct cacctggatt gagttcacag 180
ctgcctagac aggctcagac taggtgctgg gcacctggga ggaggaggag acattagcag 290
caaaggctgt taacagaagt gcctgcctag gcttggaggc aagacgctgc tgttcacagt 300
gcgagacgga ggtaggagta taatggctgt ccaggtgctg cggcagatgg tctacttcct 360
actgagtctg ttt=ctctgg tgcaaggtgc acacagtggc agcccccgag aagacttccg 420
cttctgtggc cagcggaacc agacccaaca gagcaccctc cactatgatc aatcttcaga 480
gcctcacatc tttgtgtgga acacagagga gaccctcaca attcgtgccc ccttcctggc 590
agccccagat attccccgct tcttcccaga gcctagaggg ctctatcact tctgcctcta 600
ctggagtcgc cacactggga gactccactt gcgctatggc aagcatgact acctgcttag 660
tagccaagcc tccagactcc tctgcttcca gaaacaggag cagagcctga agcagggagc 720
cccgctgatc gccacctctg tcagctcctg gcagattccc cagaacacca gcctgcctgg 780
ggctccgagc tt~atcttct ccttccacaa tgccccacac aaggtctccc acaatgcatc 890
tgtggacatg tgtgatctca agaagqaatt gcagcagctt agcaggtacc tgcagcaccc 900
Page 26 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
tcaaaaggct gccaagcc ccaccgcagc gttcatcagc cagcagttac a5 ;ctgga 960
gtcaaagctg acctctgtga gcttcctggg agacacatta tcctttgagg aggaccgggt 1020
caatgctaca gtgtggaagc tgccacccac agccggtcta gaggatctgc atatccactc 1080
ccagaaggag gaggagcaga gtgaggtcca ggcatactcg ctgttgcttc cccgggccgt 1140
attccagcag accagaggcc gtcgccggga tgacgccaag aggctcctgg tagtagactt 1200
cagcagccaa gctttgttcc aggacaagaa ttctagccaa gtcctgggtg agaaggtctt 1260
gggtattgtc gtgcagaaca ccaaagtcac caacctctca gatccggtgg tactcacctt 1320
ccagcaccag cctcagccaa aaaatgtgac tctgcagtgc gtgttctggg ttgaagaccc 1380
ggcatcaagc agcacaggga gctggagcag tgcaggctgc gagacagtga gcagagacac 1990
acagacatcc tgcctgtgca accacctgac ctactttgca gtgctgatgg tgtcatccac 1500
agaggtagaa gccactcaca aacactacct cacgctcctg tcctacgtgg gctgtgtcat 1560
ctctgctctg gcttgtgtct tcactatcgc tgcctacctc tgctccagga ggaagtcacg 1620
tgactacacc atcaaagtcc acatgaacct gctgtccgct gtcttcctgc tggacgtgag 1680
cttcctgctc agcgagcctg tggcactgac gggctccgaa gcagcctgtc gcaccagtgc 1790
catgttcctg cacttctccc tgcttgcctg cctctcctgg atgggcctcg agggctacaa 1800
tctctaccga ctggtggtgg aggtcttcgg tacctatgtg cccggctatc tgctcaagct 1860
gagcatcgtg ggctggggtt ttcctgtctt cctggtcact ctggtggcgt tggtggatgt 1920
gaataactac ggccccatta tcctagctgt gcgccggact ccggaacgtg tcacctaccc 1980
ctctatgtgc tggatccggg actccctggt gagctatgtc accaacctgg gcctcttcag 2090
tctggtgttc ctgttcaacc tggctatgct ggccaccatg gtggtgcaga tcctgcggct 2100
tcgcccgcac agccagaact ggccccacgt gctgaccctg ctgggcctca gcctggtcct 2160
tggcctcccc tgggccttgg tcttcttttc ctttgcttcc ggcaccttcc agcttgtcat 2220
cctctacctc ttcagcatca taacttccta ccaaggcttc ctcatcttcc tgtggtactg 2280
gtccatgcgg ttccaggccc aaggcggccc ctcccctctg aagaacaact cagacagcgc 2390
caaactcccc atcagctccg gcagcacctc ctccagccgc atctaagcca ccgccacacc 2900
tcccctccgg gaggacacat gcatggcgtc cgctcacgat gtctgtggcc cagtgctgtg 2460
cccacccagc ctttgttggt tagtggcata ctagagaagg ccctggtcct tgaaggcgta 2520
gggctgttgc tctggtaggt agatacctag cttgccttgg ggacgactct ggtcctcaaa 2580
gggctcagaa gcacactgcc attctgtttg tggggccgtt tcagtctgga gctaaggcct 2690
tgtctttctg gccacctctg ggtccagctg ttgctgctgg gtgttgagac ctgcagaccc 2700
aagctggggt tagatctcga aggaggctga cacatccggc ctgagacaca gctaactgtc 2760
ttgacttgct gctctgtctc tgtggtcacc atgcagatcc cgagggtggc actgggggta 2820
aatgttctgg gagaaggttt ggaggcagag caccttagga gctgagcatc tcccccagcc 2880
tttctgcaaa ccctcctctt cattccccat ccccaacccc tcctctgtgt tcccctaacc 2940
ctccacctga agcctggggt cctagaccaa tgctgtgatt tggggtggta gttcccagca 3000
gtttcctggt gccagctatc aacttctgtc tgttgtgtgg gctttggcct ctgactcagg 3060
gcaggtttct gtctgagccc tctctccaag ctgcctcacc tttgctcgca cctcagaggg 3120
acctccatct ctcctgaagc ctcctccctc tggcaagtac tgggatacag ccaccctttc 3180
aacccagcac tctgaagacc aagacagccc cctctggtga cactggccaa gcttgatctt 3240
tttcctaaga agtggtcttc agatccccgc aggtcgctca gaagacactg ggctgcctag 3300
tgtgaattct gtcctactaa cgtacagtga gcagctcctc acccccaccc ccgcaaaagc 3360
tctcaccaag tcctggagtg tcaggcaggg ggctggaaat ccaggaggac ttcctgcaaa 3920
aggcagcatt tcatcttgac ctcagccttc aggttgggga gaatgttctt tttaaatacc 3980
agttcatttg tcttttgata ttaaagctct ttatagagag tctggaaact gtaggcgatt 3590
gtcgagaaga gaaataaaaa tgagctgtta tctaatgcca tggcaaagca gcacaaaaa 3599
<210> 54
<211> 687
<212> PRT
<213> Mus musculus
<400> 59
Met Ala Val Gln Val Leu Arg Gln Met 'lal Tyr Phe _eu Leu Ser Leu
i 5 10 15
Phe Ser Leu Val Gln Gly Ala His Ser Gly Ser Prc Arg Glu Asp Phe
20 25 30
Arg Phe Cys Gly Gln Arg Asn Gln Thr Gln Gln Ser _~:r Leu His Tyr
35 40 95
Asp Gln Ser Ser Glu Pro His Ile Phe 'Ja1 Trp Asn -hr Glu Glu Thr
50 55 60
Page 27 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Leu Thr Ile Arg Ala ro Phe Leu Ala Ala Pro Asp Ile Pro x. Phe
65 70 75 80
Phe Pro Glu Pro Arg Gly Leu Tyr His Phe Cys Leu Tyr Trp Ser Arg
85 90 95
His Thr Gly Arg Leu His Leu Arg Tyr Gly Lys His Asp Tyr Leu Leu
100 105 110
Ser Ser Gln Ala Ser Arg Leu Leu Cys Phe Gln Lys Gln Glu Gln Ser
115 120 125
Leu Lys Gln Gly Ala Pro Leu Ile Ala Thr Ser Val Ser Ser Trp Gln
130 135 190
Ile Pro Gln Asn Thr Ser Leu Pro Gly Ala Pro Ser Phe Ile Phe Ser
195 150 155 160
Phe His Asn Ala Pro His Lys Val Ser His Asn Ala Ser Val Asp Met
165 170 175
Cys Asp Leu Lys Lys Glu Leu Gln Gln Leu Ser Arg Tyr Leu Gln His
180 185 190
Pro Gln Lys Ala Ala Lys Arg Pro Thr Ala Ala Phe Ile Ser Gln Gln
195 200 205
Leu Gln Ser Leu Glu Ser Lys Leu Thr Ser Val Ser Phe Leu Gly Asp
210 215 220
Thr Leu Ser Phe Glu Glu Asp Arg Val Asn Ala Thr Val Trp Lys. Leu
225 230 235 240
Pro Pro Thr Ala Gly Leu Glu Asp Leu His Ile His Ser Gln Lys Glu
295 250 255
Glu Glu Gln Ser Glu Val Gln Ala Tyr Ser Leu Leu Leu Pro Arg Ala
260 265 270
Val Phe Gln Gln Thr Arg Gly Arg Arg Arg Asp Asp Ala Lys Arg Leu
275 280 285
Leu Val Val Asp Phe Ser Ser Gln Ala Leu Phe Gln Asp Lys Asn Ser
290 295 300
Ser Gln Val Leu Gly Glu Lys Val Leu Gly Ile Val Val Gln Asn Thr
305 310 315 320
Lys Val Thr Asn Leu Ser Asp Pro Val Val Leu Thr Phe Gln His Gln
325 330 335
Pro Gln Pro Lys Asn Val Thr Leu Gln Cys Val Phe Trp Val Glu Asp
340 395 350
Pro Ala Ser Ser Ser Thr Gly Ser Trp Ser Ser Ala Gly Cys Glu Thr
355 360 365
Val Ser Arg Asp Thr Gln Thr Ser Cys Lau Cys Asn His Leu Thr Tyr
370 375 380
Phe Ala Val Leu Met Val Ser Ser Thr Glu Val Glu Ala Thr His Lys
385 390 395 400
His Tyr Leu Thr Leu Leu Ser Tyr Val Gly Cys Val .le Ser Ala Leu
Page 28 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/IJS99/19052
9C. 910 4
Ala Cys Val P'.~.e Thr Ile Ala Ala Tyr Leu Cys Ser Arg Arg Lys Ser
420 425 430
Arg Asp Tyr Ti:= Ile Lys Val His Met Asn Leu Leu Ser Ala Val Phe
435 990 495
Leu Leu Asp Vai Ser Phe Leu Leu Ser Glu Pro Val Ala Leu Thr Gly
450 455 460
Ser Glu Ala Ala Cys Arg Thr Ser Ala Met Phe Leu His Phe Ser Leu
465 970 975 980
Leu Ala Cys L2~ Ser Trp Met Gly Leu Glu Gly Tyr Asn Leu Tyr Arg
985 490 495
Leu Val Val G1~ 'Jal Phe Gly Thr Tyr Val Pro Gly Tyr Leu Leu Lys
500 505 510
Leu Ser Ile Vai Gly Trp Gly Phe Pro Val Phe Leu Val Thr Leu Val
515 520 525
Ala Leu Val Asp Val Asn Asn Tyr Gly Pro Ile Ile Leu Ala Val Arg
530 535 590
Arg Thr Pro Glu Arg Val Thr Tyr Pro Ser Met Cys Trp Ile Arg Asp
595 550 555 560
Ser Leu Val Ser Tyr Val Thr Asn Leu Gly Leu Phe Ser Leu Val Phe
565 570 575
Leu Phe Asn Leu Ala Met Leu Ala Thr Met Val Val Gln Ile Leu Arg
580 585 590
Leu Arg Pro His Ser Gln Asn Trp Pro His Val Leu Thr Leu Leu Gly
595 600 605
Leu Ser Leu Vas Leu Gly Leu Pro Trp Ala Leu Val Phe Phe Ser Phe
610 615 620
Ala Ser Gly T::r Phe Gln Leu Val Ile Leu Tyr Leu Phe Ser Ile Ile
625 630 635 640
Thr Ser Tyr Glr. Gly Phe Leu Ile Phe Leu Trp Tyr Trp Ser Met Arg
695 650 655
Phe Gln Ala Glr. Gly Gly Pro Ser Pro Leu Lys Asn Asn Ser Asp Ser
66G 665 670
Ala Lys Leu Pry Ile Ser Ser Gly Ser Thr Ser Ser Ser Arg Ile
675 680 685
<210> 55
<211> 3590
<212> DNA
<213> Mus musculus
<900> 55
ctcagggaag gcccatcttc cgggtggagg gggaagcggc gtgactggag tggaaatttt 60
tcccaacaca ac:=ctcgga ggcaacatat tggaagggac tcggggaggc cggagtccaa 120
atggaagtqg ct~aaagaaa cttctcgccc tgctgattct gagccccgcg tcgtgccgcg 180
cgccctcaat ta~~~catcg acctgtggtc ttgacagaac attcttcaca atccaaaaga 290
Page 29 of 56


CA 02340465 2001-02-21
WO 00/I 1168 PCT/US99/19052
aaaagcagac tggttcgtgtttgacgg ctatgatagc tgcagtgagg ac iiagtag'~3'00
cagctccagc tctgaggaga gtgaagaaga agttgctcc= ttaccttcca atctcccaat 360
catcaagaat aatggacaag tctacacata cccagatgg= aaatctggca tggctacctg 920
tgagatgtgt gggatggtcg gtgtgcgaga tgctttttac tctaaaacga aacgtttctg 480
cagtgtttcc tgttcaagaa gttactcgtc aaactctaag aaggcaagca ttctggccag 540
acttcagggt aagcctccaa caaagaaagc aaaagtcct= caaaaacaac ctttagttgc 600
taaattagct gcctatgccc agtatcaagc taccttgcaa aatcaagcaa agacaaaagc 660
aggcaattct gcaatctctg tggaaggctt cagctggggt aactacatca acagcaacag 720
cttcatagca gctccagtgg cctgttttaa gcatgcacct atggggacct gctggggtga 780
tatctcggaa aatgtaagga tagaagttcc caatacagac tgcagtctac ctaccaaagt 840
cttctggatt gctggaatta taaaattagc aggttataa~ gcccttttga gatatgaagg 900
atttgaaaat gattcttctc tggacttctg gtgcaatata tgtgggtctg atattcatcc 960
agttggttgg tgtgcagcta gtggaaaacc tctcgttcct cctagaactg ttcaacataa 1020
atatacaaac tggaaagctt ttctagtaaa aagacttact ggtgccaaaa cacttcctcc 1080
tgatttctca caaaaggttt ctgagagtat gcaatatcct ttcaaacctt gcatgagagt 1190
agaagtagtt gacaagaggc atttatgtcg aacaagagta gcagtggtgg aaagtgtaat 1200
tggaggacga ctacggctgg tgtatgaaga gagtgaagat ggaacagacg acttctggtg 1260
ccacatgcac agccccttaa tccaccatat tggatggtcG agaagcatag gccatcgatt 1320
caagagatca gatattacga agaaacagga cggacatttc gatacacctc cacacttatt 1380
tgctaaggta aaagaagtag accagagtgg agaatggttc aaagaaggaa tgaaattgga 1440
agctatagac ccattaaatc tttctacaat atgtgttgcc accattagaa aggtgctggc 1500
tgatggattc ctgatgattg ggattgatgg ctcagaagca gcagatggat ctgactggtt 1560
ctgttatcat gcaacctctc cttccatttt ccctgtgggt ttctgtgaaa ttaacatgat 1620
agaactgact ccacccagag gttacacaaa acttcctttt aaatggtttg actacctcag 1680
ggaaaccggc tccattgcag caccagtaaa actatttaat aaggatgttc caaaccacgg 1790
attccggtta ggaatgaaat tagaagctgt agatctcatg gagccacggt taatatgtgt 1800
agccacagtt actcgaatta ttcaccatct cttgaggata cattttgatg gttgggaaga 1860
agagtatgac cagtgqgtag actgtgagtc ccctgacctc tatcctgtag ggtggtgtca 1920
gttaactgga tatcaactac agcctccagc atcacagtca tcaagagaaa gccaatcagc 1980
ttcttcaaaa cagaagaaaa aggctaagtt tcagcaatac aaaggacata agaaaatgac 2040
cacgtcacag ctgaaggagg agctgctgga cggggaggac tatagcttcc tccatggagc 2100
atctgaccag gaaagcaatg gctctgccac cgtctacatc aaacaagagc catgaggcga 2160
ctcggaaacc atgggcaggc ggggctgttt acaggactga tttggaatca gccagctgta 2220
tagcgggcta ttctactggg acattttgct aaacacagaa aaaagttcag ttccagattt 2280
ttcaggtggg ggggaaacta ttttggtggg ggggcaattt ttcaatttat aaagacggac 2340
aatttttgtg ttgtatttga agcttttgaa agaattttgt aatattttcc aagtttggat 2400
ttatgtgcat tgttaacaag aactgaaatt ataactttt= tggtaagata aaagtttagg 2460
tagcaggatt gaaggaaatg attaagaagg atatagttgt aaatgcacat gaactgtcat 2520
tacaaatgaa ccttcttggt acctgttggg agattttttg gattttagag ttaggccagt 2580
cacatctcca gcttcctttg ctgcgaaaat atgcaactga acccctcaca gagggctcat 2690
caccatgtag atcacggaag gggtcattaa ttgtgctcgc tgacgtttta ttgcagccca 2700
tttaactgtt tgtacagaaa ctttttcatt ctgctaaaat ttatttggag ttgtatatga 2760
aactaggaga actctggata cttttatatg ctctccttca cgtaaagatg attaaaattg 2820
tctaacacta aagtgttaaa ctggaatggt tgacaaagta cacaagatct cagtctacat 2880
aaagggttgg gggaaattag tattttccta agtttattct gttttccttg ctagaaaaat 2940
cccataaaag ggatattctt gtcagacctc tgccatttct tccatctgtg agagagcaac 3000
gtgatgtaac cacacctgag agcaggtact gtcctgtgtt acaaaagaat cacatggaaa 3060
attgttaacc aaaataagtg tagatttcag aatcattcca gctctactct taactgtcct 3120
gaatttgtta gaaactgatt tgaaagaaat gtttcttaaa attccataca cacacacaca 3180
catattatga attgtcttta attgtagtca gaatttatta taacaatatt tgatttggac 3290
cattttaaac attccctatt ttaaaattca tacggcttcc ttaagaagta gaatgaaagg 3300
gtaaattggt gacatgtttg ctctctgcat tttctaacct tctaccgaat tgtgactgac 3360
tcagagagct ctagcattta ccagtgaggt tcagaaacta aactctcagg aattcattgc 3420
atctgttgta gaagtgcttc tgggtctgag cctggcctcc tcagagtggt aatactgccc 3480
acttcctctg gaaaataggc agggctaatg agaaactaat caaatgatta acctctgcgg 3540
cccttcagcc tttggaatgc taaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3590
<210> 56
<211> 631
<212> PRT
<213> Mus musculus
<400> 56
Met Phe Asp Gly Tyr Asp Ser Cys Ser Glu Asp Thr Ser Ser Ser Ser
Page 30 of 56


CA 02340465 2001-02-21
WO 00/11168 PCTNS99/19052
1 ; to
Ser Ser Glu Glu Ser Glu Glu Glu Val Ala Pro Leu Pro Ser Asn Leu
20 25 30
Pro Ile Ile Lys Asn Asn Gly Gln Val Tyr Thr Tyr Pro Asp Gly Lys
35 40 95
Ser Gly Met Ala Thr Cys Glu Met Cys Gly Met Val Gly Val Arg Asp
50 55 60
Ala Phe Tyr Ser Lys Thr Lys Arg Phe Cys Ser Val Ser Cys Ser Arg
65 70 75 80
Ser Tyr Ser Ser Asn Ser Lys Lys Ala Ser Ile Leu Ala Arg Leu Gln
85 90 95
Gly Lys Pro Pro Thr Lys Lys Ala Lys Val Leu Gln Lys Gln Pro Leu
100 105 110
Val Ala Lys Leu Ala Ala Tyr Ala Gln Tyr Gln Ala Thr Leu Gln Asn
115 120 125
Gln Ala Lys Thr Lys Ala Gly Asn Ser Ala Ile Ser Val Glu Gly Phe
130 135 140
Ser Trp Gly Asn Tyr Ile Asn Ser Asn Ser Phe Ile Ala Ala Pro Val
195 150 155 160
Ala Cys Phe Lys His Ala Pro Met Gly Thr Cys Trp Gly Asp Ile Ser
165 170 175
Glu Asn Val Arg Ile Glu Val Pro Asn Thr Asp Cys Ser Leu Pro Thr
180 185 190
Lys Val Phe Trp Ile Ala Gly Ile Ile Lys Leu Ala Gly Tyr Asn Ala
195 200 205
Leu Leu Arg Tyr Glu Gly Phe Glu Asn Asp Ser Ser Leu Asp Phe Trp
210 215 220
Cys Asn Ile Cys Gly Ser Asp Ile His Pro Val Gly Trp Cys Ala Ala
225 230 235 240
Ser Gly Lys Pro Leu Val Pro Pro Arg Thr Val Gln His Lys Tyr Thr
245 250 255
Asn Trp Lys Ala Phe Leu Val Lys Arg Leu Thr Gly Ala Lys Thr Leu
260 265 270
Pro Pro Asp Phe Ser Gln Lys Val Ser Glu Ser Met Gln Tyr Pro. Phe
275 280 285
Lys Pro Cys Met Arg Val Glu Val Val Asp Lys Arg !:is Leu Cys Arg
290 295 300
Thr Arg Val Ala Val Val Glu Ser val Ile Gly Gly Arg Leu Arg Leu
305 310 315 320
Val Tyr Glu Glu Ser Glu Asp Gly Thr Asp Asp Phe ~rp Cys His Met
325 330 335
His Ser Pro Leu Ile His His Ile Gly Trp Ser Arg Ser Ile Gly His
340 345 350
Page 31 oC56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Arg Phe Lys Arg Ser Asp Ile Thr Lys Lys Gln Asp Gly His Phe Asp
355 360 365
Thr Pro Pro His Leu Phe Ala Lys Val Lys Glu Val Asp Gln Ser Gly
370 375 380
Glu Trp Phe Lys Glu Gly Met Lys Leu Glu Ala Ile Asp Pro Leu Asn
385 390 395 400
Leu Ser Thr Ile Cys Val Ala Thr Ile Arg Lys Val Leu Ala Asp. Gly
905 910 915
Phe Leu Met Ile Gly Ile Asp Gly Ser Glu Ala Ala Asp Gly Ser Asp
420 425 430
Trp Phe Cys Tyr His Ala Thr Ser Pro Ser Ile Phe Pro Val Gly Phe
935 940 445
Cys Glu Ile Asn Met Ile Glu Leu Thr Pro Pro Arg Gly Tyr Thr Lys
950 455 960
Leu Pro Phe Lys Trp Phe Asp Tyr Leu Arg Glu Thr Gly Ser Ile Ala
965 970 975 980
Ala Pro Val Lys Leu Phe Asn Lys Asp Val Pro Asn His Gly Phe Arg
485 490 495
Val Gly Met Lys Leu Glu Ala Val Asp Leu Met Glu Pro Arg Leu Ile
500 505 510
Cys Val Ala Thr Val Thr Arg Ile Ile His Arg Leu Leu Arg Ile His
515 520 525
Phe Asp Gly Trp Glu Glu Glu Tyr Asp Gln Trp Val Asp Cys Glu Ser
530 535 540
Pro Asp Leu Asn Pro Val Gly Trp Cys Gln Leu Thr Gly Tyr Gln Leu
545 550 555 560
Gln Pro Pro Ala Ser Gln Ser Ser Arg Glu Ser Gln Ser Ala Ser Ser
565 570 575
Lys Gln Lys Lys Lys Ala Lys Ser Gln Gln Tyr Lys Gly His Lys Lys
580 585 590
Met Thr Thr Ser Gln Leu Lys Glu Glu Leu Leu Asp Gly Glu Asp Tyr
595 600 605
Ser Phe Leu His Gly Ala Ser Asp Gln Glu Ser Asn Gly Ser Ala Thr
610 615 620
Val Tyr Ile Lys Gln Glu Pro
625 630
<210> 57
<211> 1650
<212> DNA
<213> Mus musculus
<900> 57
ggggcttcac tctccagcct tggagagcag tcagggaagg cctccaggag gaagcagcta 60
cc~gtggcag ggcagagcag tgcctgtacc agtctcccgg aacttcagaa ctgcggtggc 120
Page 32 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99119052
catggagtct gtggagc~ cggtcaaaga cggcatcctc taccagcagc ac taagtt 180
tggcaagaaa tgctggcgca aagtgtgggc tctgctgtat gcgggaggcc catcaggggt 290
agctcggcta gaaagctggg acgtgcgtga tggtggcctg ggaccagcag gcgacaggtc 300
cacagggccc agccgtcgag gggaacgccg ggtcatacgc ttggctgact gtgtatctgt 360
cctgcctgcg gatggcgaga gctgtcccag ggacactggt gccttcctga ttaccaccac 420
tgagcgaagc cacctgttgg ctgcacagca ccgccagtcc tgggtggacc ccatctgtca 480
gctggccttc ccgggtaccg gagaatgttc gtcaggatca ggacaggctg agaatccaaa 590
aaggggcttt gttcccatgg aagaaaactc tatctactcc tcctggcagg aagtgaccga 600
atttccggtg atcgtgcaga agacagaagc cacctcccgc tgccagctga aaggacccta 660
cctcctggtg ctgggccaag atgacatcca actgagggag acatccaagc cccaggcctg 720
ttttagctgg ccctaccgtt tcctgcgcaa gtacggctct gacaagggtg tgttctcgtt 780
tgaggctggc cgccgctgtg actcaggtga gggccttttt gccttcagta gcccgcgtgc 840
cccagacata tgtggggttg tggctgccgc cattgcccgc cagcgggagc gtcttccaga 900
gctggccatg tccccaccct gccccctgcc tcgggccctc tccctgccct ccctagagcc 960
ccctggagag cttcgggagg tggccccagg atttgagctg cccactccca gaaagctgcc 1020
tctaactgat cccgggcctc aaagcctacc attgctgctc agccccaaca caagaaggac 1080
cggcatccgg tctctatgcg tccgtgtgca agcagaccag caagcacaca ggcacggcgg 1190
agcatttcta tgagaacgtg tgcatgctgg aggccagcct tgggctgacc aatgggggtc 1200
ctgaagccca agagggcccc cttggtggcc gcagccccct tgggcagcct tatctaccat 1260
aacactgagg atctgagttg gccgggctcg gcccaggaca gcaatctgga agcccagtac 1320
cggaggctgc tggaactgga gctggatgag gccggaagcg ccggccgctc tggagcgcag 1380
gcaggcatca aggccaagct ggtgaccctg ctgacccgtg aacggaagaa gggccccgcc 1440
ccctgtgacc ggccctgaag gcctgagcgg ccagccactg caggacagag gtgatcaccc 1500
aagaccagga acaacttcga acataacccg tctactctga cctgcaggga caagccaggt 1560
ggcccgggga ggagccacac tctgccctac ctcctccctc agactgtaca gattgaacag 1620
taataaagct tgcctatcaa cttcaaaaaa 1650
<210> 58
<211> 359
<212> PRT
<213> Mus musculus
<900> 58
Met Glu Ser Val Glu Pro Pro Val Lys Asp Gly Ile Leu Tyr Gln Gln
1 5 10 15
His Val Lys Phe Gly Lys Lys Cys Trp Arg Lys Val Trp Ala Leu Leu
20 25 30
Tyr Ala Gly Gly Pro Ser Gly Val Ala Arg Leu Glu Ser Trp Asp Val
35 90 45
Arg Asp Gly Gly Leu Gly Pro Ala Gly Asp Arg Ser Thr Gly Pro Ser
50 55 60
Arg Arg Gly Glu Arg Arg Val Ile Arg Leu Ala Asp Cys Val Ser Val
65 70 75 80
Leu Pro Ala Asp Gly Glu Ser Cys Pro Arg Asp Thr Gly Ala Phe Leu
85 90 95
Ile Thr Thr Thr Glu Arg Ser His Leu Leu Ala Ala Gln His Arg Gln
100 105 110
Ser Trp Val Asp Pro Ile Cys Gln Leu Ala Phe Pro Gly Thr Gly Glu
115 120 125
Cys Ser Ser Gly Ser Gly Gln Ala Glu Asn Pro Lys Arg Gly Phe Val
130 135 190
Pro Met Glu Glu Asn Ser Ile Tyr Ser Ser Trp Gln Glu Val Thr Glu
145 150 155 160
Phe Pro Val Ile Val Gln Lys Thr Glu Ala Thr Ser Arg Cys Gln Leu
Page 33 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
16~ 170 1
Lys Gly Pro Tyr Leu Leu Val Leu Gly Gln Asp Asp Ile Gln Leu Arg
180 185 190
Glu Thr Ser Lys Pro Gln Ala Cys Phe Ser Trp Pro Tyr Arg Phe Leu
195 200 205
Arg Lys Tyr Gly Ser Asp Lys Gly Val Phe Ser Phe Glu Ala Gly Arg
210 215 220
Arg Cys Asp Ser Gly Glu Gly Leu Phe Ala Phe Ser Ser Pro Arg Ala
225 230 235 290
Pro Asp Ile Cys Gly Val Val Ala Ala Ala Ile Ala Arg Gln Arg Glu
245 250 255
Arg Leu Pro Glu Leu Ala Met Ser Pro Pro Cys Pro Leu Pro Arg Ala
260 265 270
Leu Ser Leu Pro Ser Leu Glu Pro Pro Gly Glu Leu Arg Glu Val Ala
275 280 285
Pro Gly Phe Glu Leu Pro Thr Pro Arg Lys Leu Pro Leu Thr Asp Pro
290 295 300
Gly Pro Gln Ser Leu Pro Leu Leu Leu Ser Pro Asn Thr Arg Arg Thr
305 310 315 320
Gly Ile Arg Ser Leu Cys Val Arg Val Gln Ala Asp Gln Gln Ala His
325 330 335
Arg His Gly Gly Ala Phe Leu Glu Arg Val His Ala Gly Gly Gln Pro
340 395 350
Trp Ala Asp Gln Trp Gly Ser
355
<210> 59
<211> 1750
<212> DNA
<213> Mus musculus
<400> 59
ggcctccgac atattgcccg caggagctgc ggcggtgagc ggagagcgcc gggggaagga 60
gatgggagga cgaagaggtc ccaacaggac atcttactat cgaaacccgc tctgtgagcc 120
aggatcatca ggggcctctg gtggaggcca ctcttctagt gcatccgtga gcagcgtccg 180
ttctcgaagc aggaccactt ctgggacggg cctctccagc cctccgctgg ccgcgcagac 290
cgttgtgcct ctacagcact gcaagatccc cgagctgccc gtccaggcca gcattctgtt 300
cgagctacag ctcttcttct gtcagctcat agccctgttc gtgcactaca tcaacatcta 360
taagacagtc tggtggtatc caccctcgca cccaccctcc cacacttccc tgaacttcca 920
cctgatcgac ttcaacttgc tgatggtgac cgccattgtt ctgggccgcc gattcatcgg 980
gtccatcgtg aaggaggctt ctcagagggg gaaaggtctc cctcttccgc tccatcctgc 540
tgttcctcac ccgcttcacg ttctcacggc gacaggctgg agtctgtgcc ggtccctcat 600
ccacctcttc aggacctact ccttcctgaa cctcctgttc ctctgctatc cgtttgggat 660
gtacattccg ttcctgcagc tgaactatga tcttcgcaag acgaacctct tcacccacat 720
ggcttccatg ggaccccgag aggcagtcag tggcctggca aggagtcggg actactttct 780
gacactgcgg gagacttgga agcagcatac acgacagcta tatggcccgg aagccatgcc 890
cacccatgcc tgctgcttgt cacccagcct cattcgcaat gaagttgagt tcctcaaaat 900
ggacttcaat tggcgaatga aggaagtact tgtcagctcc atgctgagtg cctactatgt 960
ggcctttgta cctgtgtggt ttgtgaagaa tacacattac tatgacaagc gctggtcctg 1020
tgagctcttc ctgctggtgt ccatcagtac ctccgtgatc ctcatgcagc acttgttgcc 1080
tgccagctac tgtgacctgc tgcacaaggc cgccgcccac ctggcgtgct ggcagaaggt 1140
Page 34 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
ggacccagctctgtgctcatgtgc~~ca gccaccgtggactgaagagtgc 3t~gcc1200


acagggtgtgctggtgaaac acagca~gaa gcagtgggccactacaacgt1260
tgtctacaaa


ggcatcccctccgatgtctc ccactt~~gc ttttcagcaaccccctgcgg1320
ttccatttct


atcctcaacatccttttgct gctggacggt tctaccagctgtactcctta1380
gccgtcattg


atgtcctcagagaagtggca ccagaccatc tcatcctcttcagcaactac1990
tccctggccc


tatgccttcttcaagctgct ccgggac~gc gcaaagctactcctactcag1500
ctggtattgg


ccagcccccagaggacctgg accaccggtt ccagggtgatcctccaaaca1560
ctcctgagtc


acatccgggcttcacccagg ggcttcc~gg ttggggttgggggggaggga1620
ccaagggctc


tacaaaaaacaaaaacaaaa ccaccagagctttgtatttt1680
aacaacaaaa
aaaaaccaac


tgttacgtactgtttctttc tttgat~att gaaaaaagtcttatttttat1790
gatgtggtaa


actccaaaaa 1750


<210>
60


<211>
439


<212>
PRT


<213>
Mus
musculus


<400>
60


Met Gly Arg Gly Fro r.sn Tyr Tyr Asn Pro
Gly Arg Arg Thr Ser Arg


5 10 15


Leu Cys Pro Ser Ser Gly Ala Gly Gly Ser Ser
Glu Gly Ser Gly His


20 25 30


Ser Ala Val Ser Val Arg Ser Arg Thr Ser Gly
Ser Ser Arg Ser Thr


35 90 95


Thr Gly Ser Pro Pro Leu Ala Thr Val Pro Leu
Leu Ser Ala Gln Val


50 55 60


Gln His Lys Pro Glu Leu Pro Ala Ser Leu Phe
Cys Ile Val Gln Ile


65 70 75 80


Glu Leu Leu Phe Cys Gln Leu Leu Phe His Tyr
Gln Phe Ile Ala Val


85 90 95


Ile Asn Tyr Thr Val Trp 'rrpPro Ser Pro Fro
Ile Lys Tyr Pro His


10 0 105 110


Ser His Ser Asn Phe !?is Phe Asn Leu Met
Thr Leu Leu Ile Asp Leu


115 120 125


'!al Ile Leu Gly Arg Arg Gly Ser Val Lys
Thr Val Phe Ile Ile
Ala


130 135 190


Glu Ala Gln Gly Lys Gly Leu Pro Leu Pro Ala
Ser Arg Pro Leu His


145 150 155 160


Val Pro Pro His Val Leu 'rhrGly Trp Leu Cys
His Leu Ala Thr Ser


165 170 175


Arg Ser Ile Leu Phe Arg 'ChrPhe Leu Leu Leu
Leu His Tyr Ser Asn


180 185 190


Phe Leu 'ryr Phe Gly Met Tyr Phe Leu Leu Asn
Cys Pro Ile Pro Gln


195 200 205


Tyr Asp Arg Thr Asn Leu Fhe Met Ala Met Gly
Leu Lys Thr His Ser


210 215 220


Pro Arg Ala Ser Gly Leu Ala Arg Asp Phe Leu
Glu Val Arg Ser Tyr


225 230 235 240


'"hr Glt: Trp Lys Gln Hip Gln Leu Gly ro
Leu Thr Thr Arg Tyr
Arg


Page 35 of 56




CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
24; 250
Glu Ala Met Pro Thr His Ala Cys Cys Leu Ser Pro Ser Leu Ile ~.zg
260 265 270
Asn Glu Val Glu Phe Leu Lys Met Asp Phe Asn Trp Arg Met Lys ~lu
275 280 285
Val Leu Val Ser Ser Met Leu Ser Ala Tyr Tyr Val Ala Phe Val 2ro
290 295 300
Val Trp Phe Vai Lys Asn Thr His Tyr Tyr Asp Lys Arg Trp Ser ~ys
305 310 315 320
Glu Leu Phe Leu Leu Val Ser Ile Ser Thr Ser Val Ile Leu Met. ~ln
325 330 335
His Leu Leu Fro Ala Ser Tyr Cys Asp Leu Leu His Lys Ala Ala :-~la
340 345 350
His Leu Ala Cys Trp Gln Lys Val Asp Pro Ala Leu Cys Ser Asn 'lal
355 360 365
Leu Gln Pro Pzo Trp Thr Glu Glu Cys Met Trp Pro Gln Gly Val Leu
370 375 380
Val Lys His Ser Lys Asn Val Tyr Lys Ala Val Gly His Tyr Asn 'lal
385 390 395 400
Ala Ser Pro Pro Met Ser Pro Thr Ser Ala Ser Ile Ser Phe Ser Ala
405 910 415
Thr Pro Cys Gly Ser Ser Thr Ser Phe Cys Cys Trp Arg Val Pro Ser
920 425 930
Leu Ser Thr Se. Cys Thr Pro
435
<210> 61
<211> 3071
<212> DNA
<213> Mus musculus
<900> 61
gtgaaagcag cagtgcgcct ctgctccctt cagagcacag cctggtgtca aggtc~aggt 60
tccaccggct gctgctgtca ccgcagggga gtctagcccc tcccagaagg agacacagaa 120
gaatggccat ctcaactggt ttgttcctgc tgctggggct ccttggccag ccctgggcag 180
gggctgctgc tgattcacag gctgtggtgt gcgaggggac tgcctgctat acagcccatt 240
ggggcaagct gagtgccgct gaagcccagc atcgctgcaa tgagaatgga ggcaatcttg 300
ccaccgtgaa gagtgaggag gaggcccggc atgttcagca agccctgact cagctcctga 360
agaccaaggc acccttggaa gcaaagatgg gcaaattctg gatcgggctc cagcgagaga 920
agggcaactg tacgtaccat gatttgccaa tgaggggctt cagctgggtg ggtggtggag 980
aggacacagc ttattcaaac tggtacaaag ccagcaagag ctcctgtatc tttaaacgct 540
gtgtgtccct catactggac ctgtccttga cacctcaccc cagccatctg cccaagtggc 600
atgagagtcc ctgtgggacc cccgaagctc caggtaacag cattgaaggt ttcctgtgca 660
agttcaactt caaaggcatg tgtaggccac tggcgctggg tggtccaggg cgggtgacct 720
ataccacccc tttccaggcc actacctcct ctctggaggc tgtgcctttt gcctctgtag 780
ccaatgtagc tt7tggggat gaagctaaga gtgaaaccca ctatttccta tgcaatgaaa 890
agactccagg aatatttcac tggggcagct caggcccact ctgtgtcagc cccaagtttg 900
gttgcagttt caacaacggg ggctgccagc aggattgctt cgaaggtggc gatggctcct 960
tccgctgcgg ctgccggcct ggatttcgac tgctggatga tctagtaact tgtgcctcca 1020
ggaacccctg caa~tcaaac ccatgcacag gaggtggcat gtgccattct gtaccactca 1080
~tgaaaacta cac~tgccgt tgtcccagcg gctaccagct ggactctagc caagtgcact 1140
Page 36 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
gtgtggatat agatgag~. caggactccc cctgtgccca ggattgtgtc a~ ~tctag 1200
ggagcttcca c~g~gaatgt tgggttggtt accaacccag tggccccaag gaagaggcct 1260
gtgaagatgt g~atgagtgt gcagctgcca actcgccctg tgcccaaggc tgcatcaaca 1320
ctgatggctc L~~ctactgc tcctgtaaag agggctatat tgtgtctggg gaagacagta 1380
cccagtgtga g=atatagat gagtgttcgg acgcaagggg caatccatgt gattccctgt 1990
gcttcaacac agatggttcc ttcaggtgtg gctgcccgcc aggctgggag ctggctccca 1500
atggggtctt t=gtagcagg ggcactgtgt tttctgaact accagccagg cctccccaaa 1560
aggaagacaa cgatgacaga aaggagagta ctatgcctcc tactgaaatg cccagttctc 1620
ctagtggctc taaggatgtc tccaacagag cacagacaac aggtctcttc gtccaatcag 1680
atattcccac tgcctctgtt ccactagaaa tagaaatccc tagtgaagta tctgatgtct 1740
ggttcgagtt gggcacatac ctccccacga cctccggcca cagcaagccg acacatgaag 1800
attctgtgtc tgcacacagt gacaccgatg ggcagaacct gcttctgttt tacatcctgg 1860
ggacggtggt ggccatctca ctcttgctgg tgctggccct agggattctc atttatcata 1920
aacggagagc caagaaggag gagataaaag agaagaagcc tcagaatgca gccgacagct 1980
attcctgggt tccagagcga gcagagagcc aagccccgga gaatcagtac agcccaacac 2040
cagggacaga ct7ctgaaga ctatgtggcc ttagagacag ctgccactac cttcagagct 2100
accttcttag a~gaggggga agccacatca ttctgaatga cttgactgga ctctcagcaa 2160
aaaaattgtg caccttccac ttaagaacct ggtggcttgg gataggcagg tattttcttg 2220
gtgcctttga ta=3tctggg ggtgaaagct gtgtgttggt ttgtcattgt ggggagtttt 2280
gtggatattg acagacctca ctcaaacacc cttttcaaat ccaatagcaa ctggttcctc 2390
tggttcctaa ttagggggaa aggagtcaga ggggtgggac agggtggggg gatggggctt 2900
caaagttttt tcttatcact tgatttatca tcgaaggagt tactggtgct aattacaatg 2960
gaaacagttc c~~tccatca caggacagac acacctcaat cctccatggg gtcaacaact 2520
atataccccc agtgacccct taggcaagga cttgttgaga actgcatcac attttgacct 2580
gttctcaaca gtacccatct atttcaggtg ggatctctgg acctttcctc cttcccatct 2690
tgtctgcaat gtggcaaatg gcttcttttt gcatttttac tccgccccca ccccaagctg 2700
aagttcattt gcagatcagc gattaagtct gaattgtgtg gtggtcagtc ttgtttcctt 2760
ttgtcagggg ttattgtaaa tgttagtaat ttcgcctcaa gccctcagta agaacataaa 2820
tattttaaaa tatgtgcgtt tgaaatctgt ttcatgcatc ctggaactgt gggatgctca 2880
ggcaagagtg ac=ttagtct ttcagtgaat gttgcccaga atgtgggtag ggaaggctca 2990
caggttactc tcctccttag agctacaaca taacattctg aggggagtca cagggttgcc 3000
tttaaaaagt g~gagctatg tcatgctttg agctttctgt taagcacctc tcctaataaa 3060
ctctgaaaaa a 3071
<210> 62
<211> 649
<212> PRT
<213> Mus musculus
<900> 62
Met Ala Ile Ser Thr Gly Leu Phe Leu Leu Leu Gly Leu Leu Gly Gln
1 5 10 15
Pro Trp Ala G?y Ala Ala Ala Asp Ser Gln Ala Val Val Cys Glu Gly
20 25 30
Thr Ala Cys :fir Thr Ala His Trp Gly Lys Leu Ser Ala Ala Glu Ala
35 90 95
Gln His Arg Cys Asn Glu Asn Gly Gly Asn Leu Ala Thr Val Lys Ser
50 55 60
Glu Glu Glu ~:~_a Arg His Val Gln Gln Ala Leu Thr Gln Leu Leu Lys
65 70 75 80
Thr Lys Ala F=o Leu Glu Ala Lys Met Gly Lys Phe Trp Ile Gly Leu
85 90 95
Gln Arg Glu ~;~s Gly Asn Cys Thr Tyr His Asp Leu Pro Met Arg Gly
100 105 110
Phe Ser Trp '.'al Gly Gly Gly Glu Asp Thr Ala Tyr Ser Asn Trp Tyr
115 120 125
Page 37 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Lys Ala Ser Lys Se. 2r Cys =.e Phe Lys Ar? Cys Val Ser I Ile
130 135 140
Leu Asp Leu Ser Leu Thr Pro His Pro Ser His Leu Pro Lys Trp His
145 150 155 160
Glu Ser Pro Cys Gly Thr Pro Glu Ala Pro G1~ Asn Ser Ile Glu Gly
165 170 175
Fhe Leu Cys Lys Phe Asn Phe Lys Gly Met Cys Arg Pro Leu Ala Leu
180 185 190
Gly Gly Pro Gly Arg Val Thr :'yr Thr Thr Prc Phe Gln Ala Thr Thr
195 200 205
Ser Ser Leu Glu Ala Val Pro Fhe Ala Ser Val Ala Asn Val Ala Cys
210 215 220
Gly Asp Glu Ala Lys Ser Glu Thr His Tyr Phe Leu Cys Asn Glu Lys
225 230 235 24p
Thr Pro Gly Ile Phe His Trp Gly Ser Ser Gly Pro Leu Cys Val Ser
295 250 255
Fro Lys Phe Gly Cys Ser Phe Asn Asn Gly Gly Cys Gln Gln Asp Cys
260 265 27p
Fhe Glu Gly Gly Asp Gly Ser Phe Arg Cys Gly Cys Arg Pro Gly Phe
275 280 285
Arg Leu Leu Asp Asp Leu Val Thr Cys Ala Ser Arg Asn Pro Cys Ser
290 295 300
Ser Asn Pro Cys Thr Gly Gly Gly Met Cys His Ser Val Pro Leu Ser
305 310 315 320
Giu Asn Tyr Thr Cys Arg Cys Fro Ser Gly Tyr Gln Leu Asp Ser Ser
325 330 335
Gln Val His Cys Val Asp Ile Asp Glu Cys Gln Asp Ser Pro Cys Ala
390 395 350
Gln Asp Cys Val Asn Thr Leu Gly Ser Phe His Cys Glu Cys Trp Val
355 360 365
Gly Tyr Gln Pro Ser Gly Pro Lys Glu Glu Ala Cys Glu Asp Val Asp
370 375 380
Glu Cys Ala Ala Ala Asn Ser Pro Cys Ala Gln Gly Cys Ile Asn Thr
X85 390 395 400
Asp Gly Ser Phe Tyr Cys Ser Cys Lys Glu Gly Tyr Ile Val Ser Gly
905 910 915
Glu Asp Ser Thr Gln Cys Glu Asp Ile Asp Glu Cys Ser Asp Ala Arg
920 925 930
Giy Asn Pro Cys Asp Ser Leu Cys Phe Asn Thr Asp ~ly Ser Phe Arg
935 490 ;q5
Cys Gly Cys Pro Pro Gly Trp Glu Leu Ala Fro Asn ~iy Val Phe Cys
95D 455 460
Ser Arg Gly Thr Val Phe Ser Glu Leu Fro Ala Arg ,ro Pro Gln Lys
Page 38 of 56


CA 02340465 2001-02-21
WO 00/11168 PCTNS99/19052
465 70 975
Glu Asp Asn Asp Asp Arg Lys Glu Ser Thr Met Pro Pro Thr Glu :!et
985 990 995
Pro Ser Ser Pro Ser Gly Ser Lys Asp Val Ser Asn Arg Ala Gln .hr
500 505 510
Thr Gly Leu Phe Val Gln Ser Asp Ile Pro Thr Ala Ser Val Pro Leu
515 520 525
Glu Ile Glu Ile Pro Ser Glu Val Ser Asp Val Trp Phe Glu Leu aly
530 535 590
Thr Tyr Leu Pro Thr Thr Ser Gly His Ser Lys Pro Thr His Glu asp
595 550 555 ,60
Ser Val Ser Ala His Ser Asp '"hr Asp Gly Gln Asn Leu Leu Leu the
565 570 575
Tyr Ile Leu Gly Thr Val Val Ala Ile Ser Leu Leu Leu Val Leu i~la
580 585 590
Leu Gly Ile Leu Ile Tyr His Lys Arg Arg Ala Lys Lys Glu Glu ile
595 600 605
Lys Glu Lys Lys Pro Gln Asn Ala Ala Asp Ser Tyr Ser Trp Val ?ro
610 615 620
Glu Arg Ala Glu Ser Gln Ala Pro Glu Asn Gln Tyr Ser Pro Thr Pro
625 630 635 690
Gly Thr Asp Cys
<210> 63
<211> 999
<212> DNA
<213> Mus musculus
<900> 63
cccattactt ccacctccca tgaggactgc acctgcggca gcgattttat gtgaacttgg 60
attactgtat ggaaatggag gagtgagaaa gtgtgggaat cataaaagaa ggcagaacca 120
aaagacagag ggatcttggg ccatgagtca cagcaaggaa agcctcccac caaacatctg 180
cactggactc ctacaggaac aagaaatgaa ctatcgtgtt cagcccctga gactt~ggct 290
taacatgatt ttcacattgt ctaccctacc taatagagca gagatgtaaa tattattctt 300
attttagagg tgtgatgcct cagctgcaat gggtgagaac tactcctcat ttattctctt 360
ccaaggcaat aaaagagaat ggaccaaaga cagtctgtca tcacatctag tcaaaagagc 920
taatgtcgca gtacaactct tcaaaagaaa aagaaaaaac aagaaaaaag taataaacag 980
atgtgttctg cttgaaaaa qgg
<210> 69
<211> 89
<212> PRT
<213> Mus musculus
<900> 64
Met Arg Thr Ala Pro Ala Ala Ala Ile Leu Cys Glu ~eu Gly Leu ~eu
1 5 10 15
Tyr Gly Asn Gly Gly Val Arg Lys Cys Gly Asn His ys Arg Arg ~ln
20 25 30
Page 39 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Asn Gln Lys T::r G?u Gly Ser Try Ala Met Ser His Ser Lys Glu Ser
35 40 45
Leu Pro Pro Asn .le Cys Thr Gly Leu Leu Gln Glu Gln Glu Met Asn
50 55 60
Tyr Arg Val Gln ?ro Leu Arg Leu Gly Leu Asn Met Ile Phe Thr Lee:
65 70 75 8C
Ser Thr Leu Pro As.~. Arg Ala Glu Met
'~ 5
<210> 65
<211> 3857
<212> DNA
<213> Mus muscul;a
<900> 65
agagggtaga cagaaaggcg ggaaagggct gtgaggtcaa atggacccca tggaactgaa 60
caacgtcagc atcgaacccg acggagacag ctgcagcggg gacagtattc aggacagcta 120
caccggcatg gaaaactccg acaaggacgc catgaacagc caatttgcta atgaagatgc 180
cgaaagtcag aagttcctga caaatgggtt tttagggaag aagaagctag ccgattacgc 290
ggatgagcat caccctggaa tgacttcctt tggaatgtcc tcatttaacc tgagcaacgc 300
catcatgggc agtgggatct taggcttgtc ctatgccatg gccaacaccg ggatcatcct 360
ttttataatc atgctgct~a ctgtggcaat actctcgctc tactcggttc accttttgct 420
gaagacagcc aaggaaggag ggtctctaat ctatgaaaaa ttgggcgaga aagcatttgg 480
atggcctggg aaaattggag ccttcatctc tattacaatg cagaacattg gagccatgtc 590
aagctacctc ttcatcatta agtacgaact gcctgaagta atcagagcat tcatgggact 600
tgaagaaaac actggggaat ggtacctcaa cggcaactac ctcgtcttat ttgtgtcc~t 660
ggggatcatc ctcccgctct ctctccttaa aaatttaggc taccttggct acaccagtgg 720
attttctctc tcctgcatgg tgtttttcgt cagtgtggtg atttacaaaa aattccaaat 780
tccctgccct ctgcc~cctc tggatcacaa caacggaaat ctgacgttca acaacacact 840
tccgattcac atgatc;ccc tgcctaatga ctcggagagc tcgggtgtga acttcatgat 900
ggattacgct caccacaacc cagctgggct ggatgagaag caggtcgcag gccctctt~a 960
cagcaatggc gtggag=acg aagcccaggg tgctgagaaa tgccaaccaa aatactt=y~ 1020
gttcaattcc cggacg7cct atgcaatccc aatcctggct tttgcttttg tctgccaccc 1080
tgaggtcctt cccatc=aca gcgagcttaa agatcgatcc cgcagaaaga tgcagacgy~ 1140
gtccaacatt tccatc~cag g~a=3ctcgt ca=gtacctt cttgcggccc tctttggt=a 1200
tctgagcttc tacggggacg ttcaagacga gctgctgcat gcttacagca aggtctaca~ 1260
atttgatacg gctcttctca tggtgcgcct ggcagtcctg gtggcagtga cactgaccgt 1320
gcccatcgtg ctgttcccya tcc;=acttc ggtgatcaca ctgctgtttc caaggaaac~ 1380
cttcagctgg ctgaagca_~ tcgggatcgc tgcaatcatc atcgcactca acaacatcc= 1990
ggtcatcctc gtgcctacca tcaaatacat ctttggattc ataggggctt cttctgccac 1500
tatgctgatt ttcattc:;c cggctgcgtt ttatctcaag ctcgtcaaga aagaacctct 1560
aagatcaccc cagaaaa~:g gggctttggt cttccttgtg actggaatta ttttcatgat 1620
gggaagcatg gcgctcat~a tactcgactg gatctacaac ccgccgaatc ccaatcacca 1680
ctaat~ccgg ggagacgc.;t ctccactgga aacagctgaa attgtctgaa ggacattt=a 1790
gttg=cttga ttggga=yet agtctgagga attagcaaga ttccaaagac gtttttctag 1800
ctctatcagc acacatttta acccaggccg tgcagtgcag tgtgtgatgc ccgagttgtg 1860
tttgcagcag ctgtgcaayc tgaagcctgt tggctgcgtg tgttggtcag cagacaatag 1920
cctg=ccccc catggtcac~ ccacttctct ccacccccag attaacaggt aattctactc 1980
tcagaacatc agacaaagac ctcctggttg ggatacttgt ggaagagaaa attatgggtt 2090
ttgttgggaa tggtttt7tt gggaatggtg aaggatgcat taaaaattct gtgcgaagta 2100
tcatcagtta cggccatc_c tcactctaca ccaacactaa gggtcggttg actagctgag 2160
gcagggggat atcttgg;~t gtccctgtga ggatcatgac gtatgacggt tgccagtata 2220
gagtac~tca tttcaa~~_= caaggaatag tttgcccaac ctgcttatta caccgagtta 2280
gtgaat;ttc attct~=_=a ttt~ttggta tggcaaatgt tcaaacatct cactaaacat 2340
agaggggggt tattta~~_= atcatgaaac aactaccaaa aagtaatggt ttttaaca~c 2900
tgccttttca tgttgtttct gatatctttg ttccactttg tctttgaaca aggcttcccc 2960
tctcg=cttc cgttct~a~t ct~cgtaat~ ttcagaaaaa ccacagtccg tgtgggagac 2520
acactacccc agtatt;ttt gatacatctc tatttgataa acattcagtg caggaaac_g 2580
tgattttgct atatgtt:7t gtacataatc tcattctgca gttatcagaa cgtt.gacata 2690
tgggacattg gatttt=a~= ttttacatat g~aggttttt tttcttcaca gacaaaatg= 2700
Page 40 of 36


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
ttatatcatc agggtggggc agggaattaa gctggtgggc tcaaaaatcc atgggtactt 2760
atctgtccat tggagacatc taaaaattaa agtcaaagtt gtgcatagtt cagtaatgct 2820
cttcactgtt tacaagacta taatcatctc agcaaagtag cgaaggaagg gtttgtaaat 2880
aattttcagt gctaacaatg gtctttgaat ttaacatgtc tagaataggg tttagttcat 2990
tttagtttac atcggaactt ggtgacattc atttgccatt aatagaacat cgtgtggtta 3000
ggaataaatg caccaacatg acccaaaacc acattgctca catccatgga cgcttacacg 3060
ggatggagtt cagactccgc ccactgccta gaatctctgg cattgacttc tttttttcaa 3120
ttactggttg tatattttac atttcagtaa aaagccaagt ctgaaatgca agtatcctac 3180
aggtagtgat atactgtcac accaaagcag tttttcaaca gtgtgcctaa gaaacatata 3290
tttgtttact gtgtcatttt ctggacctta agactataca atggtgtgct ttgtgataga 3300
aattgagcat tactagaaac taccataact tcggagttat tatcatttgg gagggaaaaa 3360
aatcaccaaa taggatcttt aagatgtctt tctattttca ttcatccaag aaataaatca 3920
agatagaaag cagaaagggt gtgctgcaaa tattaaatta ttattttatc tgaacatatt 3980
aatgtgaata taaaagatta atccaaaaat attggaaacc tgtgaaatct gtacgtattt 3590
cggatcttta cacttagagc agctaacctg gaagtatcca cttgtgtagt tagatattca 3600
agagcaatat cacagtgcca aagacttcat tcacacagca ccgatcattt gtgtggccgt 3660
tttcaggatt agagttcaca cagactccag ggcagatact cagaataccg ttttctggta 3720
aatttaaccc atttgtaaac agatacaaat tttattttct tacatcattt ataagacggt 3780
tcaatgtact ggggggattt tttttattac agactgtgta ttggtatata ataaataaac 3840
ttttcaaatg aaaaaaa 3857
<210> 66
<211> 547
<212> PRT
<213> Mus musculus
<900> 66
Met Asp Pro Met Glu Leu Asn Asn Val Ser Ile Glu Pro Asp Gly Asp
I 5 10 15'
Ser Cys Ser Gly Asp Ser I'_e Gln Asp Ser Tyr Thr Gly Met Glu Asn
20 25 30
Ser Asp Lys Asp Ala Met Asn Ser Gln Phe Ala Asn Glu Asp Ala Glu
35 40 45
Ser Gln Lys Phe Leu Thr As: Gly Phe Leu Gly Lys Lys Lys Leu Ala
50 5~ 60
Asp Tyr Ala Asp Glu His H_s Pro Gly Met Thr Ser Phe Gly Met Ser
65 70 75 80
Ser P~.e Asn Leu Ser Asn A1a Ile Met Gly Ser Gly Ile Leu Gly Leu
85 90 g5
Ser Tyr Ala Met Ala Asn Thr Gly Ile Ile Leu Phe Ile Ile Met Leu
100 105 110
Leu Thr Val Ala Ile Leu Se. Leu Tyr Ser Val His Leu Leu Leu Lys
115 120 I25
Thr Ala Lys Glu Gly Gly Se_- Leu Ile Tyr Glu Lys Leu Gly Glu Lys
130 13~ 190
Ala Phe Gly Trp Pro Gly Lys Ile Gly Ala Phe Ile Ser Ile Thr Met
195 150 155 i60
Gln Asn Ile Gly Ala Met Se= Ser Tyr Leu Phe Ile Ile Lys Tyr Glu
165 170 I75
Leu ?_-o Glu Val Ile Arg Ala Phe Met Gly Leu Glu Glu Asn Thr Gly
180 185 190
Glu Trp Tyr Leu Asn Gly Asn Tyr Leu Val Leu Phe Val Ser Val Gly
Page 4 t of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
195 200 205
Ile Ile Leu Pro Leu Ser Leu Leu Lys Asn Leu Gly Tyr Leu Gly Tyr
210 215 220
Thr Ser Gly Phe Ser Leu Ser Cys Met Val Phe Phe Val Ser Val Val
225 230 235 240
Ile Tyr Lys Lys Phe Gln Ile Pro Cys Pro Leu Pro Ala Leu Asp His
295 250 255
is n Asn Gly Asn Leu Thr Phe Asn Asn Thr Leu Pro Ile His Met Ile
260 265 270
Ser Leu Pro Asn Asp Ser Glu Ser Ser Gly Val Asn Phe Met Met Asp
275 280 285
Tyr Ala His His Asn Pro Ala Gly Leu Asp Glu Lys Gln Val Ala Gly
290 295 300
Pro Leu His Ser Asn Gly Val Glu Tyr Glu Ala Gln Gly Ala Glu Lys
305 310 315 320
Cys Gln Pro Lys Tyr Phe Val Phe Asn Ser Arg Thr Ala Tyr Ala Ile
325 330 335
Pro Ile Leu Ala Phe Ala Phe Val Cys His Pro Glu Val Leu Pro Ile
390 395 350
Tyr Ser Glu Leu Lys Asp Arg Ser Arg Arg Lys Met Gln Thr Val Ser
355 360 365
Asn Ile Ser Ile Ser Gly Met Leu Val Met Tyr Leu Leu Ala Ala Leu
370 375 380
Phe Gly Tyr Leu Ser Phe Tyr Gly Asp Val Glu Asp Glu Leu Leu His
385 390 395 900
rla Tyr Ser Lys Val Tyr Thr Phe Asp Thr Ala Leu Leu Met Val Arg
905 410 915
Leu Ala Val Leu Val Ala Val Thr Leu Thr Val Pro Ile Val Leu Phe
920 425 930
Pro Ile Arg Thr Ser Val Ile Thr Leu Leu Phe Pro Arg Lys Pro Phe
435 990 495
Ser Trp Leu Lys His Phe Gly Ile Ala Ala Ile Ile Ile Ala Leu Asn
450 955 960
Asn Ile Leu Val Ile Leu Val Pro Thr Ile Lys Tyr Ile Phe Gly Phe
965 470 975 980
Ile Gly Ala Ser Ser Ala Thr Met Leu Ile Phe Ile Leu Pro Ala Ala
485 990 995
Phe Tyr Leu Lys Leu Val Lys Lys Glu Pro Leu Arg Ser Pro Gln Lys
500 505 510
Ile Gly Ala Leu Val Phe Leu Val Thr Gly Ile Ile Phe Met Met Gly
515 520 525
Ser Met Ala Leu Ile Ile Leu Asp Trp Ile Tyr Asn Pro Pro Asn Pro
530 535 59G
Page 42 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Asn His His
595
<210> 67
<211> 1070
<212> DNA
<213> Mus musculus
<900> 67
agcgacatgg ccccgcccgc gctccaggcc cagcctccag gcggctctca actgaggttc 60
ctgctgttcc tgctgctgtt gctgctgctg ctgtcatggc catcgcaggg ggacgccctg 120
gcaatgcctg aacagcgacc ctccggccct gagtcccaac tcaacgccga cgagctacgg 180
ggtcgcttcc aggacctgct gagccggctg catgccaacc agagccgaga ggactcgaac 240
tcagaaccaa gtcctgaccc agctgtccgg atactcagtc cagagqtgag attggggtcc 300
cacggccagc tgctactccg cgtcaaccgg gcgtcgctga gtcagggtct ccccgaagcc 360
taccgcgtgc accgagcgct gctcctgctg acgccgacgg cccgcccctg ggacatcact 420
aggcccctga agcgtgcgct cagcctccgg ggaccccgtg ctcccgcatt acgcctgcgc 480
ctgacgccgc ctccggacct ggctatgctg ccctctggcg gcacgcagct ggaactgcgc 540
ttacgggtag ccgccggcag ggggcgccga agcgcgcatg cgcacccaag agactcgtgc 600
ccactgggtc cggggcgctg ctgtcacttg gagactgtgc aggcaactct tgaagacttg 660
ggctggagcg actgggtgct gtccccgcgc cagctgcagc tgagcatgtg cgtgggcgag 720
tgtccccacc tgtatcgctc cgcgaacacg catgcgcaga tcaaagcacg cctgcatggc 780
ctgcagcctg acaaggtgcc tgccccgtgc tgtgtcccct ccagctacac cccggtggtt 840
cttatgcaca ggacagacag tggtgtgtca ctgcagactt atgatgacct ggtggcccgg 900
ggctgccact gcgcttgagc accgggccct gctcctcacc tacactcccc ttcaaggatg 960
ctatttatat ttgtatttat taatattatt aatttattgg ggtcgggctg ggtggatgga 1020
ttgtgtattt atttaaaact ctgctaataa aggtgagctt ggtttcaaaa 1070
<210> 68
<211> 298
<212> PRT
<213> Mus musculus
<400> 68
Met Ala Pro Pro Ala Leu Gln Ala Gln Pro Pro Gly Gly Ser Gln Leu
1 5 10 15
Arg Phe Leu Leu Phe Leu Leu Leu Leu Leu Leu Leu Leu Ser Trp Fro
20 25 30
5er Gln Gly Asp Ala Leu Ala Met Pro Glu Gln Arg Pro Ser Gly Fro
35 40 95
Glu Sex Gln Leu Asn Ala Asp Glu Leu Arg Gly Arg Phe Gln Asp Leu
50 55 60
Leu Ser Arg Leu His Ala Asn Gln Ser Arg Glu Asp Ser Asn Ser Glu
65 70 75 80
Pro Ser Pro Asp Pro Ala Val Arg Ile Leu Ser Pro Glu Val Arg Leu
85 90 95
Gly Ser His Gly Gln Leu Leu Leu Arg Val Asn Arg Ala Ser Leu Ser
100 105 110
Gln Gly Leu Pro Glu Ala Tyr Arg Val His Arg Ala Leu Leu Leu ~eu
115 120 125
Thr Pro Thr Ala Arg Pro Trp Asp Ile Thr Arg Pro Leu Lys Arg ~la
130 135 140
:.eu Ser Leu Arg Gly Pro Arg Ala Pro Ala Leu Arg ieu Arg Leu hr
Page 43 of 56


CA 02340465 2001-02-21
WO 00/11168 PCTNS99/19052
195 50 155 .60
Pro Pro Pro Asp Leu Ala Met Leu Pro Ser Gly Gly Thr Gln Leu ~~u
165 170 175
Leu Arg Leu Arg Val Ala Ala Gly Arg Gly Arg Arg Ser Ala His .la
180 185 190
His Pro Arg Asp Ser Cys Pro Leu Gly Pro Gly Arg Cys Cys His _eu
195 200 205
Glu Thr Val Gin Ala Thr Leu Glu Asp Leu Gly Trp Ser Asp Trp '.'al
210 215 220
Leu Ser Pro Arg Gln Leu Gln Leu Ser Met Cys Val Gly Glu Cys =ro
225 230 235 290
His Leu Tyr A_g Ser Ala Asn Thr His Ala Gln Ile Lys Ala Arg _au
245 250 255
His Gly Leu Gln Pro Asp Lys Val Pro Ala Pro Cys Cys Val Pro Ser
260 265 270
Ser Tyr Thr Pro Val Val Leu Met His Arg Thr Asp Ser Gly Val Ser
275 280 285
Leu Gln Thr Tyr Asp Asp Leu Val Ala Arg
290 295
<210> 69
<211> 134
<212> PRT
<213> Mus musculus
<400> 69
Ala Lys Tyr Va_ Cys Leu Ala Asn Lys Asn Cys Pro Val Asp Lys :-.rg
1 5 10 15
Arg Arg Asn Arg Cys Gln Tyr Cys Arg Phe Gln Lys Cys Leu Ala '.'al
20 25 30
Gly Met Val Lys Glu Val Val Arg Thr Asp Ser Leu Lys Gly Arg Arg
35 90 95
Gly Arg Leu Pro Ser Lys Pro Lys Ser Pro Gln Glu Pro Ser Pro Pro
50 55 60
Ser Pro Pro Phe Gln Ala Asn Pro Asp Tyr Gln Met Ser Gly Asp nsp
65 70 75 80
Thr Gln His I~.:e Gln Gln Phe Tyr Asp Leu Leu Thr Gly Ser Met Flu
85 90 95
Ile Ile Arg Gay Trp Ala Glu Lys Ile Pro Gly Phe Ala Asp Leu ro
1C0 105 110
Lys Ala Asp G:.n Asp Leu Leu Phe Glu Ser Ala Phe Leu Glu Leu .he
115 120 125
Val Leu Arg Leu Ala Tyr
130
Page 44 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
<210> 70
<211> 190
<212> PRT
<213> Mus musculus
<900> 70
Ser Gln Thr Arg Gln Gln Gly Pro Leu Arg Ser Ile Met Lys Asp Leu
1 5 10 15
His Ser Asp Asp Asn Glu Glu Glu Ser Asp Glu Val Glu Asp Asn Asp
20 25 30
Asn Asp Ser Glu Met Glu Arg Pro Val Asn Arg Gly Xaa Ser Arg Ser
35 90 95
Arg Arg Val Ser Leu Ser Asp Gly Ser Asp Ser Glu Ser Ser Ser Ala
50 55 60
Ser Ser Pro Leu His His Glu Pro Pro Pro Pro Leu Leu Lys Thr Asn
65 70 75 80
Asn Asn Gln Ile Leu Glu Val Lys Ser Pro Ile Lys Gln Ser Lys Ser
B5 90 95
Asp Lys Gln Ile Lys Asn Gly Glu Cys Asp Lys Ala Tyr Leu Asp Glu
100 105 110
Leu Val Glu Leu Pro Arg Xaa Xaa Met Thr Leu Arg Glu Arg His Ile
115 120 125
Leu Gln Gln Ile Val Asn Leu Xaa Glu Glu Thr Gly
130 135 190
<210> 71
<211> 89
<212> PRT
<213> Mus musculus
<900> 71
Met Arg Thr Ala Pro Ala Ala Ala Ile Leu Cys Glu Leu Gly Leu Leu
1 5 10 15
Tyr Gly Asn Gly Gly Val Arg Lys Cys Gly Asn His Lys Arg Arg Gln
20 25 30
Asn Gln Lys Thr Glu Gly Ser Trp Ala Met Ser His Ser Lys Glu Ser
35 90 45
Leu Pro Pro Asn Ile Cys Thr Gly Leu Leu Gln Glu Gln Glu Met Asn
50 55 60
Tyr Arg Val Gln Pro Leu Arg Leu Gly Leu Asn Met Ile Phe Thr Leu
65 70 75 80
Ser Thr Leu Pro Asn Arg Ala Glu Met
85
<210> 72
<211> 3121
<212> DNA
<213> Homo sapiens
Page 45 of 56


CA 02340465 2001-02-21
WO 00/11168 PC'TlUS99/19052
<400> 72
gaaagcagca gtgcgcctct gctcccttca gagcacagcc tggtgtcaag gtccaggttc 60
caccggctgc tgctgtcacc gcaggggagt ctagcccctc ccagaaggag acacagaaga 120
atggccatct caactggttt gttcctgctg ctggqgctcc ttggccagcc ctgggcaggg 180
gctgctgctg attcacaggc tgtggtgtgc gaggggactg cctgctatac agcccattgg 290
ggcaagctga gtgccgctga agcccagcat cgctgcaatg agaatggagg caatcttgcc 300
accgtgaaga gtgaggagga ggcccggcat gttcagcaag ccctgactca gctcctgaag 360
accaaggcac ccttggaagc aaagatgggc aaattctgga tcgggctcca gcgagagaag 920
ggcaactgta cgtaccatga tttgccaatg aggggcttca gctgggtggg tggtggagag 480
gacacagctt attcaaactg gtacaaagcc agcaagagct cctgtatctt taaacgctgt 540
gtgtccctca tactggacct gtccttgaca cctcacccca gccatctgcc caagtggcat 600
gagagtccct gtgggacccc cgaagctcca ggtaacagca ttgaaggttt cctgtgcaag 660
ttcaacttca aaggcatgtg taggccactg gcgctgggtg gtccagggcg ggtgacctat 720
accacccctt tccaggccac tacctcctct ctggaggctg tgccttttgc ctctgtagcc 780
aatgtagctt gtggggatga agctaagagt gaaacccact atttcctatg caatgaaaag 890
actccaggaa tatttcactg gggcagctca ggcccactct gtgtcagccc caagtttggt 900
tgcagtttca acaacggggg ctgccagcag gattgcttcg aaggtggcga tggctccttc 960
cgctgcggct gccggcctgg atttcgactg ctggatgatc tagtaacttg tgcctccagg 1020
aacccctgca gctcaaaccc atgcacagga ggtggcatgt gccattctgt accactcagt 1080
gaaaactaca cttgccgttg tcccagcggc taccagctgg actctagcca agtgcactgt 1190
gtggatatag atgagtgcca ggactccccc tgtgcccagg attgtgtcaa cactctaggg 1200
agcttccact gtgaatgttg ggttggttac caacccagtg gccccaagga agaggcctgt 1260
gaagatgtgg atgagtgtgc agctgccaac tcgccctgtg cccaaggctg catcaacact 1320
gatggctctt tctactgctc ctgtaaagag ggctatattg tgtctgggga agacagtacc 1380
cagtgtgagg atatagatga gtgttcggac gcaaggggca atccatgtga ttccctgtgc 1440
ttcaacacag atggttcctt caggtgtggc tgcccgccag gctgggagct ggctcccaat 1500
ggggtctttt gtagcagggg cactgtgttt tctgaactac cagccaggcc tccccaaaag 1560
gaagacaacg atgacagaaa ggagagtact atgcctccta ctgaaatgcc cagttctcct 1620
agtggctcta aggatgtctc caacagagca cagacaacag gtctcttcgt ccaatcagat 1680
attcccactg cctctgttcc actagaaata gaaatcccta gtgaagtatc tgatgtctgg 1740
ttcgagttgg gcacatacct ccccacgacc tccggccaca gcaagccgac acatgaagat 1800
tctgtgtctg cacacagtga caccgatggg cagaacctgc ttctgtttta catcctgggg 1860
acggtggtgg ccatctcact cttgctggtg ctggccctag ggattctcat ttatcataaa 1920
cggagagcca agaaggagga gataaaagag aagaagcctc agaatgcagc cgacagctat 1980
tcctgggttc cagagcgagc agagagccaa gccccggaga atcagtacag cccaacacca 2090
gggacagact gctgaagact atgtggcctt agagacagct gccactacct tcagagctac 2100
cttcttagat gagggggaag ccacatcatt ctgaatgact tgactggact ctcagcaaaa 2160
aaattgtgca ccttccactt aagaacctgg tggcttggga taggcaggta ttttcttggt 2220
gcctttgata tgtctggggg tgaaagctgt gtgttggttt gtcattgtgg ggagttttgt 2280
ggatattgac agacctcact caaacaccct tttcaaatcc aatagcaact ggttcctctg 2390
gttcctaatt agggggaaag gagtcagagg ggtgggacag ggtgggggga tggggcttca 2900
aagttttttc ttatcacttg atttatcatc gaaggagtta ctggtgctaa ttacaatgga 2960
aacagttcct ttccatcaca ggacagacac acctcaatcc tccatggggt caacaactat 2520
atacccccag tgacccctta ggcaaggact tgttgagaac tgcatcacat tttgacctgt 2580
tctcaacagt acccatctat ttcaggtggg atctctggac ctttcctcct tcccatcttg 2690
tctgcaatgt ggcaaatggc ttctttttgc atttttactc cgcccccacc ccaagctgaa 2700
gttcatttgc agatcagcga ttaagtctga attgtgtggt ggtcagtctt gtttcctttt 2760
gtcaggggtt attgtaaatg ttagtaattt cgcctcaagc cctcagtaag aacataaata 2820
ttttaaaata tgtgcgtttg aaatctgttt catgcatcct ggaactgtgg gatgctcagg 2880
caagagtgac tttagtcttt cagtgaatgt tgcccagaat gtgggtaggg aaggctcaca 2990
ggttactctc ctccttagag ctacaacata acattctgag gggagtcaca gggttgcctt 3000
taaaaagtgg gagctatgtc atgctttgag ctttctgtta agcacctetc ctaataaact 3060
ctgaaaaaat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120
a 3121
<210> 73
<211> 644
<212> PRT
<213> Homo sapiens
<400> 73
Met Ala Ile Ser Thr Gly Leu Phe Leu Leu Leu Gly ~eu Leu Gly Gln
i 5 10 15
Page 46 of S6


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Pro Trp Ala Gly Al la Ala Asp Ser Gln Ala Val Val Cys C Cly
20 25 30
Thr Ala Cys Tyr Thr Ala His Trp Gly Lys Leu Ser Ala Ala Glu ~la
35 90 95
Gln His Arg Cys Asn Glu Asn Gly Gly Asn Leu Ala Thr Val Lys Ser
50 55 60
Glu Glu Glu Ala Arg His Val Gln Gln Ala Leu Thr Gln Leu Leu Lys
65 70 75 80
Thr Lys Ala Pro Leu Glu Ala Lys Met Gly Lys Phe Trp Ile Gly Leu
85 90 95
Gln Arg Glu Lys Gly Asn Cys Thr Tyr His Asp Leu Pro Met Arg Giy
100 105 110
Phe Ser Trp Val Gly Gly Gly Glu Asp Thr Ala Tyr Ser Asn Trp =yr
115 120 125
Lys Ala Ser Lys Sex Ser Cys Ile Phe Lys Arg Cys Val Ser Leu Ile
130 135 190
Leu Asp Leu Ser Leu Thr Pro His Pro Ser His Leu Pro Lys Trp His
195 150 155 160
Glu Ser Pro Cys Gly Thr Pro Glu Ala Pro Gly Asn Ser Ile Glu Gly
165 170 175
Phe Leu Cys Lys Phe Asn Phe Lys Gly Met Cys Arg Pro Leu Ala Leu
180 185 190
Gly Gly Pro Gly Arg Val Thr Tyr Thr Thr Pro Phe Gln Ala Thr Thr
195 200 205
Ser Ser Leu Glu Ala Val Pro Phe Ala Ser Val Ala Asn Val Ala Cys
210 215 220
Gly Asp Glu Ala Lys Ser Glu Thr His Tyr Phe Leu Cys Asn Glu Lys
225 230 235 290
Thr Pro Gly Ile Phe His Trp Gly Ser Ser Gly Pro Leu Cys Val Ser
245 250 255
Pro Lys Phe Gly Cys Ser Phe Asn Asn Gly Gly Cys Gln Gln Asp Cys
260 265 270
Phe Glu Gly Gly Asp Gly Ser Phe Arg Cys Gly Cys Arg Pro Gly Phe
275 280 285
Arg Leu Leu Asp Asp Leu Val Thr Cys Ala Ser Arg Asn Pro Cys Ser
290 295 300
Ser Asn Pro Cys Thr Gly Gly Gly Met Cys His Ser Val Pro Leu Ser
305 310 315 320
Glu Asn Tyr Thr Cys Arg Cys Pro Ser Gly Tyr Gln Leu Asp Ser Ser
325 330 335
Gln Val His Cys Val Asp Ile Asp Glu Cys Gln Asp Ser Pro Cys Fla
340 395 350
Gln Asp Cys Val Asn Thr Leu Gly Ser Phe His Cys Glu Cys Trp '!al
Page 47 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
355 360 365
Gly Tyr Gln Pro Ser Gly Pro Lys Glu Glu Ala Cys Glu Asp Val hsp
370 375 380
Glu Cys Ala Ala Ala Asn Ser Pro Cys Ala Gln Gly Cys Ile Asn Thr
385 390 395 400
Asp Gly Ser Phe Tyr Cys Ser Cys Lys Glu Gly Tyr Ile Val Ser Gly
905 910 915
Glu Asp Ser T!:r Gln Cys Glu Asp Ile Asp Glu Cys Ser Asp Ala Arg
920 925 430
Gly Asn Pro Cys Asp Ser Leu Cys Phe Asn Thr Asp Gly Ser Phe Arg
435 990 495
Cys Gly Cys P=c Pro Gly Trp Glu Leu Ala Pro Asn Gly Val Phe Cys
950 955 960
Ser Arg Gly T::r Val Phe Ser Glu Leu Pro Ala Arg Pro Pro Gln Lys
965 970 475 480
Glu Asp Asn Asp Asp Arg Lys Glu Ser Thr Met Pro Pro Thr Glu Met
485 490 995
Pro Ser Ser Prc Ser Gly Ser Lys Asp Val Ser Asn Arg Ala Gln Thr
500 505 510
Thr Gly Leu Phe Val Gln Ser Asp Ile Pro Thr Ala Ser Val Pro Leu
515 520 525
Glu Ile Glu Ile Pro Ser Glu Val Ser Asp Val Trp Phe Glu Leu Gly
530 535 590
Thr Tyr Leu P=~ Thr Thr Ser Gly His Ser Lys Pro Thr His Glu Asp
545 550 555 560
Ser Val Ser Ala His Ser Asp Thr Asp Gly Gln Rsn Leu Leu Leu Phe
565 570 575
Tyr Ile Leu Gi; Thr Val Val Ala Ile Ser Leu Leu Leu Val Leu Ala
580 585 590
Leu Gly Ile Le~~ Ile Tyr His Lys Arg Arg Ala Lys Lys Glu Glu Ile
595 600 605
Lys Glu Lys Lys Pro Gln Asn Ala Ala Asp Ser Tyr Ser Trp Val Pro
610 615 620
Glu Arg Ala G_~a Ser Gln Ala Pro Glu Asn Gln Tyr Ser Pro Thr Pro
625 630 635 690
Gly Thr Asp Cys
<210> 79
<211> 27
<212> DNA
<2I3> Artific;al Sequence
<220>
<223> Descrip~=o~ of Artificial Sequence: Primer
Page 48 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
<400> 79


tggtgtcgac gcagagtagc 27
ggccgct


<210> 75


<211> 19


<212> DNA


<213> Artificial Sequence


<220>


<223> Description of Sequence: Primer
Artificial


<900> 75


ggcccgggcc ggcc 14


<210> 76


<211> 17


<212> DNA


<213> Artificial Sequence


<220>


<223> Description of Sequence: Primer
Artificial


<400> 76


tcgaggccgc ccgggcc 17


<210> 77


<211> 18


<212> DNA


<213> Artificial Sequence


<220>


<223> Description of Sequence: Primer
Artificial


<400> 77


cagccctcac tccttctc 18


<210> 78


<211> 18


<212> DNA


<213> Artificial Sequence


<220>


<223> Description of Sequence: Primer
Artificial


<400> 78


ggtggggtct ttcattcc 18


<210> 79


<211> 18


<212> DNA


<213> Artificial Sequence


<220>


<223> Description of Sequence: Primer
Artificial


<400> 79


ttcagcaagc cctgactc 18


<210> 80


<211> 18


<212> DNA


<213> Artificial Sequence


Page 49 of Sb


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
<2zo>
<223> Description of Artific_a= Sequence: Primer
<900> 80
gccaccttcg aagcaatc 18
<210> 81
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> B1
gagcggtaca ggagaatg 18
<210> 82
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 82
gcccacccaa ccaaatca . 18
<210> 83
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<900> 83
accgcgttct tctgtaac 18
<210> 89
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<900> 89
cagctaacag caggatcc 18
<210> 85
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<900> $5
gacccaggct gcccc 15
<210> 86
<211> 15
<212> DNA
Page 50 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
<213> Artificial S ence
<220>


<223> Description of Sequence:Primer
Artificial


<400> 86


ggtactcttg ttgag 15


<210> 87


<211> 39


<212> DNA


<213> Artificial Sequence


<220>


<223> Description of Sequence:Primer
Artificial


<400> 87


gagggtacca aa~=tcgtgg gaag 34
tccgcaacca


<210> 88


<211> 36


<212> DNA


<213> Artificial Sequence


<220>


<223> Description of Sequence:Primer
Artificial


<400> 88


ctcagatctg aattcggcct gaggag 36
gtccctcggt


<210> 89


<211> 35


<212> DNA


<213> Artificial Sequence


<z2o>


<223> Description of Sequence:Primer
Artificial


<400> 89


gacaagatct cagctgaata ctgtg 35
gcgacccacc


<210> 90


<211> 41


<212> DNA


<213> Artificial Sequence


<220>


<223> Description of Sequence:Primer
Artificial


<900> 90


gcatctagag cggccgctca tcggtgagga 91
ggcctgtccc g


<210> 91


<211> 37


<212> DNA


<213> Artificial Sequence


<220>


<223> Descript'_on of Sequence:Primer
Artificial


<400> 91


gcatctagag cggccgcgaa atctcct 37
cgctgcttag


<210> 92
Page 51 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/IJS99/19052
<211> 131
<212> PRT
<213> Mus musculus
<900> 92
Glu Val Phe Leu Asn Pro Leu Gly Arg Asp Leu Leu Ser Ile Ser Asp
1 5 10 15
Gly Arg Xaa Arg Ala Ile His Arg Arg Gly His Asn Asp Gly Glu Asp
20 25 30
Ser Leu Thr His Thr Asp Val Ser Ser Phe Gln Thr Met Asp Gln Met
35 90 95
Val Ser Asn Met Arg Asn Tyr Met Gln Lys Leu Glu Arg Asn Thr Gly
50 55 60
Gln Leu Ser Val Asp Pro Asn Gly His Ser Phe Cys Ser Ser Ser Val
65 70 75 BO
Met Thr Tyr Ser Lys Ile Gly Asp Glu Pro Pro Lys Val Phe His Ala
85 90 95
Ser Thr Gln Thr Arg Arg Ala Pro Gly Arg Ile Lys Glu Thr Arg Lys
100 105 110
Ala Met Arg Asp Ser Asp Ser Gly Thr Arg Lys His Gly Tyr Trp Ser
115 120 125
Ser Tyr Pro
130
<210> 93
<211> 131
<212> PRT
<213> Mus musculus
<400> 93
Arg Ser Phe Ser Glu Pro Phe Gly Arg Asp Leu Leu Ser Ile Ser Asp
1 5 10 15'
Gly Arg Gly Arg Ala His Asn Arg Arg Gly His Asn Asp Gly Glu Asp
20 25 30
Ser Leu Thr His Thr Asp Val Ser Ser Phe Gln Thr Met Asp Gln Met
35 90 45
Val Ser Asn Met Arg Asn Tyr Met Gln Lys Leu Glu Arg Asn Phe Gly
SO 55 60
Gln Leu Ser Val Asp Pro Asn Gly His Ser Phe Cys Ser Ser Ser Val
65 70 75 80
Met Thr Tyr Ser Lys Ile Gly Asp Glu Pro Pro Lys Val Phe Gln Ala
85 90 95
Ser Thr Gln Thr Rrg Arg Ala Pro Gly Gly Ile Lys Glu Thr Arg Lys
100 105 I10
Ala Met Arg Asp Ser Asp Ser Gly Leu Glu Lys Met Ala Ile Gly His
115 120 125
His Ile His
Page 52 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
130
<210> 94
<211> 159
<212> PRT
<213> Mus musculus
<900> 99
Ala Lys Tyr Val Cys Leu Ala Asn Lys Asn Cys Pro Val Asp Lys Arg
1 5 10 15
Arg Arg Asn Arg Cys Gln Tyr Cys Arg Phe Gln Lys Cys Leu Ala Val
20 25 30
Gly Met Val Lys Glu Xaa Val Arg Thr Asp Ser Leu Lys Gly Arg Arg
35 90 95
Gly Arg Leu Pro Ser Lys Pro Lys Ser Pro Gln Glu Pro Ser Pro Pro
50 55 60
Ser Pro Pro Val Ser Leu Ile Ser Ala Leu Val Arg Ala His Val Asp
65 70 75 80
Ser Asn Pro Ala Met Thr Ser Leu Asp Tyr Ser Arg Phe Gln Ala Asn
85 90 95
Pro Asp Tyr Gln Met Ser Gly Asp Asp Thr Gln His Ile Gln Gln Phe
100 105 110
Tyr Asp Leu Leu Thr Gly Ser Met Glu Ile Ile Arg Gly Trp Ala Xaa
115 120 125
Xaa Ile Pro Gly Phe Ala Asp Leu Pro Lys Ala Asp Gln Asp Leu Leu
130 135 190
Phe Glu Ser Ala Phe Leu Glu Leu Phe Val Leu Azg Leu Ala Tyr
195 150 155
<210> 95
<211> 140
<212> PRT
<213> Mus musculus
<900> 95
Ser Gln Thr Arg Gln Gln Gly Pro Leu Arg Ser Ile Met Lys Asp Leu
1 5 10 15'
His Ser Asp Asp Asn Glu Glu Glu Ser Asp Glu Val Glu Asp Asn Asp
20 25 30
Asn Asp Ser Glu Met Glu Arg Pro Val Asn Arg Gly Gly Ser Arg Ser
35 90 45
Arg Arg Val Ser Leu Ser Asp Gly Ser Asp Ser Glu Ser Ser Ser Ala
50 55 60
Ser Ser Pro Leu His His Glu Pro Pro Pro Pro Leu Leu Lys Thr Asn
65 70 75 80
Asn Asn Gln Ile Leu Glu Val Lys Ser Pro Ile Lys Gln Ser Lys Ser
85 90 95
Page 53 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Asp Lys Gln Ile Ly_ sn Gly G";u Cys Asp Lys Ala Tyr Leu . Glu
100 105 110
Leu Val Glu Leu His Arg Arg :.eu Met Thr Leu Arg Glu Arg His Ile
115 120 125
Leu Gln Gln Ile Val Asn Leu Ile Glu Glu Thr Gly
130 135 190
<210> 96
<211> 652
<212> PRT
<213> Mus musculus
<900> 96
Met Ala Thr Ser Met Gly Leu Leu Leu Leu Leu Leu Leu Leu Leu Thr
1 5 10 15
Gln Pro Gly Ala Gly Thr Gly Ala Asp Thr Glu Ala Val Val Cys Val
20 25 30
Gly Thr Ala Cys Tyr Thr Ala His Ser Gly Lys Leu Ser Ala Ala Glu
35 40 45
Ala Gln Asn His Cys Asn Gln Asn Gly Gly Asn Leu Ala Thr Val Lys
50 55 60
Ser Lys Glu Glu Ala Gln His Val Gln Arg Val Leu Ala Gln Leu Leu
65 70 75 80
Arg Arg Glu Ala Ala Leu Thr Ala Arg Met Ser Lys Phe Trp Ile Gly
85 90 95
Leu Gln Arg Glu Lys Gly Lys Cys Leu Asp Pro Ser Leu Pro Leu Lys
100 105 110
Gly Phe Ser Trp Val Gly Gly Gly Glu Asp Thr Pro Tyr Ser Asn Trp
115 120 125
His Lys Glu Leu Arg Asn Ser Cys Ile Ser Lys Arg Cys Val Ser Leu
130 135 190
Leu Leu Asp Leu Ser Gln Pro Leu Leu Pro Asn Arg Leu Pro Lys Trp
195 150 155 160
Ser Glu Gly Pro Cys Gly Ser Pro Gly Ser Pro Gly Ser Asn Ile Glu
165 170 175
Gly Phe Val Cys Lys Phe Ser Phe Lys Gly Met Cys Arg Pro Leu Ala
180 185 190
Leu Gly Gly Pro Gly Gln Val Thr Tyr Thr Thr Pro Phe Gln Thr Thr
195 200 205
Ser Ser Ser Leu Glu Ala Val Pro Phe Ala Ser Ala Ala Asn Val~ Ala
210 215 220
Cys Gly Glu Gly Asp Lys Asp Glu Thr Gln Ser His Tyr Phe Leu Cys
225 230 235 240
Lys Glu Lys Ala Pro Asp Val Phe Asp Trp Gly Ser Ser Gly Pro Leu
295 250 255
Page 54 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
Cys Val Ser Pro Ly. yr Gly Cys Asn Phe Asn Asn Gly Gly ~ :iis
260 265 270
Gln Asp Cys Phe Glu Gly Gly Asp Gly Ser Phe Leu Cys Gly Cys Arg
275 280 285
Pro Gly Arg Arg Leu Leu Asp Asp Leu Val Thr Cys Ala Ser Arg Asn
290 295 300
Pro Cys Ser Ser Ser Pro Cys Arg Gly Gly Ala Thr Cys Val Leu Gly
305 310 315 320
Pro His Gly Lys Asn Tyr Thr Cys Arg Cys Pro Gln Gly Tyr Gln Leu
325 330 335
Asp Ser Ser G'_n Leu Asp Cys Val Asp Val Asp Glu Cys Gln Asp Ser
340 395 350
Pro Cys Ala Glz Glu Cys Val Asn Thr Pro Gly Gly Phe Arg Cys Glu
355 360 365
Cys Trp Val Gly Tyr Glu Pro Gly Gly Pro Gly Glu Gly Ala Cys Gln
370 375 380
Asp Val Asp Glu Cys Ala Leu Gly Arg Ser Pro Cys Ala Gln Gly Cys
385 390 395 900
Thr Asn Thr Asp Gly Ser Phe His Cys Ser Cys Glu Glu Gly Tyr Val
905 410 415
Leu Ala Gly Glu Asp Gly Thr Gln Cys Gln Asp Val Asp Glu Cys Val
420 925 430
Gly Pro Gly Gly Pro Leu Cys Asp Ser Leu Cys Phe Asn Thr Gln Gly
935 490 995
Ser Phe His Cys Gly Cys Leu Pro Gly Trp Val Leu Ala Pro Asn Gly
450 955 460
Val Ser Cys Thr Met Gly Pro Val Ser Leu Gly Pro Pro Ser Gly Pro
465 970 975 480
Pro Asp Glu Glu Asp Lys Gly Glu Lys Glu Gly Ser Thr Val Pro Arg
485 990 995
Ala Ala Thr Ala Ser Pro Thr Arg Gly Pro Glu Gly Thr Pro Lys Ala
500 505 510
Thr Pro Thr T':r Ser Arg Pro Ser Leu Ser Ser Asp Ala Pro Ile Thr
515 520 525
Ser Ala Pro Leu Lys Met Leu Ala Pro Ser Gly Ser Ser Gly Val Trp
530 535 540
Arg Glu Pro Ser Ile His His Ala Thr Ala Ala Ser Gly Pro Gln Glu
545 550 555 560
Pro Ala Gly G'_y Asp Ser Ser Val Ala Thr Gln Asn Asn Asp Gly Thr
565 570 575
Asp Gly Gln Lvs Leu Leu Leu Phe Tyr Ile Leu Gly Thr Val Val nla
580 585 590
-_le Leu Leu Te;.i Leu Ala Leu Ala Leu Gly Leu Leu Val Tyr Arg Lys
Page 55 of 56


CA 02340465 2001-02-21
WO 00/11168 PCT/US99/19052
595 600 6'05"'
Arg Arg Ala ys Arg Glu Glu Lys Lys Glu Lys Lys Pro Gln Asn Ala
610 615 620
Ala Asp Ser Tyr Ser Trp Val Pro Glu Arg Ala Glu Ser Arg Ala Met
625 630 635 690
Glu Asn Gln Tyr Ser Pro Thr Pro Gly Thr Asp Cys
645 650
Page 56 of 56