Note: Descriptions are shown in the official language in which they were submitted.
w o 92/06116 Z ~'6 9 q 4 6 Pcr/ussl/o70s3
HYBRID GROWTH FACTORS
~ACKGROUND OF THE ~NVENTION
Within this application several publications are referenced by Arabic
numerals within parentheses. Full citations for these references may
be found at the end of the specification immediately preceding the
Sequence Listing. The disclosures of these publications in their
entirety are hereby incorporated by reference into this application in
order to more fully describe the state of the art to which this
invention pertains.
A variety of factors can influence the activity of a cell. Frequently
a factor exerts its influence by interacting with a receptor on the
surface of a cell. After binding to the receptor, the signal which
determines the cellular response to the factor can be mediated through
a number of different events, including internalization of the factor
or alterations of the receptor caused by ligand binding. During the
course of hematopoietic differentiation, a number of different factors
are involved in the maturation of a pluripotent stem cell into a fully
d1fferent1ated cell. The activities of these factors during the
course of hematopoietic differentiat10n have resulted in these factors
belng chartcter1zed as early factors or late factors. For example,
factors such ~s 1nterleuktn-3 (IL-3) and granulocyte-macrophage colony
st1mulat1ng factor ~GM-CSF) are cons1dered early factors, while
erythropoietln (Epo), macrophage colony stlmulating factor (M-CSF),
and granulocyte colony st1mulat1ng factor (G-CSF) are considered late
factors.
8ased upon stud1es performed with purified factors and in vitro colony
fo~m1ng unit assays, it appears that both IL-3 and GM-CSF act on
pluripotent cells before they become committed to a particular
hematopo1et1c pathway. After the events stimulated by these factors
are underway, such lineage restr1cted cells become recept1ve to
3S further d1fferent1ation mediated by such late factors as Epo, (wh1ch
leads to the maturat10n of erythrocytes), G-CSF (which leads cells
'.''. ' `'-'"'~',.'' '.' ;' ' ''. '
, , . ,. , . . ....... .. .- . . ... . .- ... . ~ . . - . .
. . ~ . . .. - . ~ . .. ... . . . . - - -
WO 92/06116 PCI/I.S91/07053
2 C ~ 9 ~ ~ 6 ~ -- . t ~
-- 2
into the granulocytic pathway), and M-CSF (which leads to the
maturation of macrophages). Experiments described in recent
publications (1,2,3) have demonstrated in vitro that early or late
factors alone are poor stimuli of colony formation. However, when an
early factor such as IL-3 or GM-CSF is combined with a late factor,
levels of colony formation equivalent to that seen with conditioned
media having full activity is observed. Thus, differentiation appears
to be dependent upon the dual activities of early and late factors.
lo Despite a clear r~t:quirement for both IL-3 or GM-CSF and Epo for the
formation of erythroid colony forming units, published results
indicate that IL-~ can down-modulate high affinity Epo receptors (4).
Because the amount of IL-3 required to demonstrate down-modulation of
the Epo receptor was higher than that reported by others who
lS demonstrated functional full IL-3 activity in the presence of Epo, it
is unclear whether this phenomenon is relevant m vivo.
Previous experiments in animals (22-26) suggest that under conditions
of hematopoietic regeneration, optimal expansion of late progenitors
could only occur in the presence of an adequate early progenitor pool.
This then makes manipulations that result in the expansion of early
hematopoietlc progenitor pools extremely desirable. IL-3 has been
shown to exert a differentiat~ve and proliferative effect on early
progenltor cells and at IL-3 concentratlons which had little or no
effect alone, Epo acted synergistically to 1nduce proliferat10n and
d1fferentlatlon of erythrold progen~tors. (27) By targettng a
molecule wlth both early (IL-3) and late (Epo and G-CSF) activitles to
early progenltor cells, opt~mal expansion of a desired lineage should
be posslble.
WO 92/061 16 ` - - . PCI'/US91/07053
~ 2~69746;
-- 3
SUMMARY OF THE INVENTION
The present invention concerns hybrid mo1ecules comprising early and
late differentiation factors produced by genetic manipulation. By
covalently linking such factors the local concentration of -the late
factor is very high at the surface of a cell to which the early factor
is bound. Additionally, if down-modul~tion is relevant 1n vivo,
binding of late factors to any remaining low-affinity receptors, e.g.
Epo receptors, could be enhanced, thus reducing the amount of late
lo factor required to stimulate the cell. Furthermore, by linking an
early factor with a late factor, such early factor may act more
specifically to stimulate only the desired lineage, thus reducing any
undesirable effects mediated by the early factor. Finally, it is
considerably easier to produce and administer to a patient a single
factor with two activities rather it would to produce and administer
two separate factors.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a ~estern blot analysis of IL-3/Epo hybrid growth
factors in CHO CM. CHO CM was collected from clones 23-10 (IL-3:Epo
Flex), 5-4 (IL-3:Epo Short) and 17-3-1 (Epo:lL-3 ShortJ. Hybrid
growth factor concentrations were determined by ELISA assay. CM
contaln~ng 74 ng of IL-3:Epo Flex (having a 23 aa flexible linker
(lane 2), 73.5 ng of IL-3:Epo Short (having a short 2 aa 11nker) (lane
3), 80 ng of Epo:lL-3 Short (hav~ng a 3 aa linker) (lane 4 ) were
sub~ected to SDS-PAGE (10-20X gel) electrophoresis and were assayed
for Epo by ~estern blotting with a mouse anti-Epo polyclonal ant1sera
as descr~bed in Example 7. Medium conditioned by CHO cells
transfected with the vector pEe6 (lane 5) and rHu Epo 10 ng (lane 6),
20 ng (lane 7), 30 ng (lane 8), 70 ng (lane 9), and 100 ng (lane lO)
were included. Molecular s1ze markers in kilodaltons (lane 1).
F1gure 2 shows AMLl93 cells proliferate in response to the IL-3
moiety of the hybr~d growth factors. AML193 cells were grown to
stat10nary phase and suspended in RPMI-1640 plus 10% FCS and growth
WO 92/06116 2(~9746 PCl/US91/07053
factor deprived for 16 hours. The indicated concentrations of growth
factors were added for 42 hours followed by a 6 hour pulse of (3H)
thymidine as described in Example 7. N0 GF (no growth factor); CH0
CM (medium conditioned by CH0 cells transfected with the vector pEe6)i
Epo (rHu Epo); IL-3 (rHu IL-3); IL-3:Epo Flex (CH0 CM containing IL-
3:Epo fusion protein with a 23aa flexible linker); IL-3:Epo Short (CH0
CM containing IL-3:Epo fusion protein with a 2aa linker); Epo:lL-3
Short (CH0 CM containing Epo:lL-3 fusion protein with a 3aa linker)~
Figure 3 shows dose response of IL-3 adapted AMLI93 cells to the IL-3
moiety of the hybrid growth factors. IL-3 adapted AML 193 cells were
grown to stationary phase and suspended in RPMI-1640 plus I0% FCS
minus growth factor for 16 hours. Increasing concentrations of IL-3
and fusion proteins were added and the assay was carried out as
described in Figure 2 and in Example 7. IL-3:Epo Flex (CH0 CM
containing IL-3:Epo fusion protein with a 23aa flexible linker); IL-
3:Epo Short (CH0 CM containing IL-3:Epo fusion protein with a 2aa
linker); Epo:lL-3 Short (CHO CM containing Epo:lL-3 fusion protein
with a 3aa linker).
Figure 4 shows FDC-PI/ER cells proliferate in response to the Epo
mo1ety of the hybrid growth factors. FDC-PI/ER cells were grown to
stat1Onary phase and suspended in RPMI-1640 plus 10% FCS without
growth factor for 16 hours. The 1nd1cated concentrations of growth
factors were added for 42 hours followed by a 6 hour pulse of ( H)
thymldlne as descrlbed 1n Example 7. Columns are labeled as
descr1bed 1n F1gure 2. ~EH13 CM (med1um cond1tioned by murine ~EHI3
cells which produce and secrete IL-3).
Figure 5 shows dose response of FDC-PI/ER cells to the Epo moiety of
the hybr1d growth factors. FDC-PI/ER cells were grown to stat1Onary
phase and suspended in RPMI-1640 plus IOX FCS and deprived of growth
factor for I6 hours. Increasing concentrations of Epo and fusion
prote1ns were added and the assay was carried out as described in
3S Example 7. Hybrid growth factors are as designated in Figure 3.
- .: ;. , , . . .. .; . . , , . , : .~, .. . : . - .
-, - . , - .. . , , , . ~ :. . : . -
. . : . . .. . - : ..
, , . - . . . - . .. . - . . . .
- .. . . . - -... .. : ..... : .. .
- ~, . , . . . . .. .. . - - ... ..
WO 92/06116 2~697a~6 PCr/lJS91/07053
::
Figure 6 shows IL-3 plus Epo responsiveness of IL-3 adapted TF-l
cells. TF-I cells adapted for growth in IL-3 were grown to stationary
phase and suspended in RPMI-1640 plus 10% FCS minus growth factor for
16 hours. Increasing concentrations of growth factors, 0.75 fmol/ml
hybrid growth factors and 0.75 fmol/ml Epo plus 1.5 fmol/ml IL-3 (1),
1.5 fmol/ml hybrid growth factors and 1.5 fmol/ml of Epo plus 3.0
fmol/ml IL-3 (2), 3.0 fmol/ml hybrid growth factors and 3.0 fmol/ml
Epo plus 6.0 fmol/ml IL-3 (3)1 were added and the assay was carried
out as described in Example 7. Hybrid growth factors are as
designated in Figure 3.
Figure 7 shows dose responsiveness of IL-3 adapted TF-l cells to the
hybrid growth factors. TF-1 cells adapted for growth in IL-3 were
grown to log phase and suspended in RPMI-1640 plus 10% FCS minus
growth factor for 16 hours. Increasing concentrations of hybrid
growth factors were added and the cells were incubated for 8 hours.
(3H) Thymidine (1 ~Ci/well) was added and the incubation was continued
for 16 hours. (A) Dose response to hybrid growth factor,
concentrations of 0 to 30 fmol/ml. (B) Represents the same data as in
A for concentrations of 0 to 1.875 fmol/ml to emphasize the
differences between hybrid factors. Hybrid growth factors are as
designated in Figure 3.
Figure 8 shows dose responsiveness of GM-CSF adapted TF-I cells to the
2S hybr1d growth factors,- TF-l cells mainta~ned in GM-CSF were grown to
log phase and suspended in RPMI-1640 plus 10X FCS minus growth factor
for 16 hours. Increasing concentrat1Ons of hybrid growth factors were
added and the assay was carried out as described above for figure 5.
(A) Dose response to hybrid growth factor ccncentrations, of 0 to 30
fmol/ml. (B) Represents the same data as in A for concentrations of
0 to 1.875 fmol/ml to emphasize the differences between hybrid
factors. Hybrid growth factors are as designated in figure 3.
F~gure 9 shows TF-I cells proliferate in response to the IL-3 moiety
3S of the IL-3/G-CSF hybrid growth factor. Tf-l cells were grown to
stationary phase and suspended in RPMI-1640 plus IOX FCS deprived of
- ' '
.:
' . ' ., , ", , ' ~., : ~ .` .. . "." ' . . . :
2~97~6
WO 92/06116 - PCr/US91/07053
6 --
growth factor for l6 hours. The indicated concentrations of growth
factors were added and the assay was carried out as described in
Example 7. Factors are as designated in Figure 2 except, G-CSF (rHu
G-CSF); IL-3/G-CSF (CHO CM containing IL-3/G-CSF fusion protein with
S a IOaa linker).
Figure lO shows NFS-60 cells proliferate in response to the G-CSF
moiety of the IL-3,~G-CSF hybrid growth factor. NFS-60 cells were
grown to stationar~ phase and suspended in RPMI-1640 plus l0% FCS
lo minus growth factor for l6 hours. The indicated co~centrations of
growth factors were added and the assay was carried out as described
in Example 7. Growth factors are as designated in Figures 2 and 9.
Figure ll shows dose responslveness of AMLI93 cells to the iL-3:G-CSF
hybrid growth factor. AMLl93 cells were grown to stationary phase and
suspended in RPMI-1640 plus l0% FCS deprived of growth factor for l6
hours. The indicated concentrations of growth factors were added and
the assay was carried out as described in Example 7. Growth factors
are as designated in Figures 2 and 9.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a recombinant hematoyoietic molecule
comprising at least a port10n of a first hematopoietic molecule having
2i5 early myelo1d dlfferent1at10n activity and at least a portion of a
second h~m-topo1et1c molecule having late myelo1d differentiation
act1v1ty. Th1s retomb1nant molecule has early myeloid differentiation
act1vity associated with the first hematopoietic molecule and late
myelo1d dlfferentiat10n activity associated with the second
hematopoiet1c molecule. Within this application, "hematopo1etlc
molecule~ means a molecule wh1ch promotes and/or regulates
hematopoiesis. Hematopoietic molecules exert such promotional or
regulatory activities at different stages during hematopoiesis, such
stages being referred to herein as early myeloid differentiation and
3S late myeloid different1ation. Also within th1s application, ~early
myelo1d differentiation activity" means the ability to promote the
WO 92/06116 PCI/US91/0~053
~ Z~69746
-- 7
differentiation, self-renewal, or proliferation of pluripotent ~yeloid
cells, i.e., stem cells or colony forming unit,
granulocyte-erythrocyte-monocyte-megacaryocyte, cells. Moreover,
within this application, "late myeloid differentiation activity~ means
s the ability to promote the maturation or differentiation of a lineage
restricted myeloid cell, i.e., a myeloid precursor cell committed to
a specific cell lineage such as erythrocytes, megakaryocytes,
monocytes, neutrophils, eosinophils, and basophils.
In one embodiment of the invention, the first hematopoietic 001ecule
is selected from the group consisting of IL-3 and GM-CSF. In another
embodiment of the invention, the second hemopoietic molecule is
selected from the group consisting of Epo, G-CSF, IL-5 and M-CSF. In
a preferred embodiment of the invention, the portion of the first
hematopoietic molecule is linked to the portion of the second
hematopoietic molecule by an amino acid linker sequence comprising at
least two amino acid residues.
~ithin the context of the present invention, it is understood that
variations in proteins and nucleic acids exist among individuals, e.g.
amino acid or nucleotide substitutions, deletions, insertions, and
degree or location of glycosylation, and that func~ional derivatives
resulting therefrom are included within the scope of the present
lnvent10n.
In a preferred embodiment of the invention, the recombinant molecule
comprises the ent1re amino acid sequence of human IL-3 (SEQ ID N0: I).
Moreover, the recombinant hematopoietic molecule may preferably
comprise a 79 amino acid sequence derived from human IE-3 (SE3 ID N0:
2),i.e~ residues 1-79 of SEQ ID N0: I.
Further still, in yet another preferred embodiment of the invention,
the recombinant molecule comprises the entire amino acid sequence of
human erythropoietin (SEQ ID N0: 3). In still a further embodiment of
the invention, the hemopoietic molecule comprises a I55 amino acid
WO 92/06116 . ! ~ `. '' ` '' ,"'. PCI/US91/07053
C, . ,-
-- 8
sequence derived from human erythropoietin (SEQ ID N0: 4), i.e.,
residues 7-16l of SEQ ID N0: 3.
In another preferred embodiment of the invention, the recombinant
hematopoietic molecule comprises the entire amino acid sequence of
human G-CSF (sEq ID N0: 5).
In one embodiment of the invention, the first hematopoietic molecule
is IL-3 and the second hematopoietic molecule is erythropoietin. The
lo first hematopoietic molecule~ i.e. IL-3, may comprise the amino
portion and the second hematopoietic molecule~ i.e. Epo, may comprise
the carboxyl portion of the recombinant molecule. Preferably, the
recombinant hematopoietic molecule comprises the amino acid sequence
from amino acid 1 to amino acid 302 of SE~ ID N0: 6. Also preferably,
the recombinant hematopoietic molecule comprises the amino acid
sequence from amino acid I to amino acid 321 of SEQ ID N0: 7.
However, in another embodiment of the invention, the first
hematopoietic molecule, i.e. IL-3, may comprise the carboxyl portion
and the second hemopoietic molecule, i.e. Epo, may comprise the amino
portion of the recombinant molecule. In a preferred embodiment of the
1nvention, the recombinant molecule comprises the amino acid sequence
from amino acid I to amino acid 303 of SEQ ID N0: 8. In yet another
preferred embodiment, the recombinant molecule comprises the amino
ac1d sequence from amino acid 1 to amlno acid 322 of SEQ ID N0: 9.
2S
In still a further embodiment of the invention, the first
hematopoietic molecu1e ts IL-3 and the second hematopoietic molecule
ls G~CSF. In one such embodiment, the firsl hematopoietic molecu1e
comprises the amino portion and the second hematopoietic molecule
comprises the carboxyl portion of the recombinant molecule. In yet a
more specific embodiment, the recombinant molecule comprises the amino
acid sequence from amino acid 1 to amino acid 317 of SEQ ID N0: 10.
The subject invention also provides nucleic acid molecules which
3S encode the recombinant hematopoietic molecules of the subject
invention. Examples of such nucleic acid molecules are SEQ ID N0: 11,
.. : . ~ . . . , . . .. . .: . . .
- .. . -. ~
- . .
, ': :, :;, , :' ' ' - ;.; ' , ,. ~ . . . . .
.. . . . , , . .. . .. . , . : . . .
w o 92/06116 Zl!1i9746 pcT/ussl/o7os3
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
Moreover. vectors which comprise the nucleic acid molecules of the
subject invention are also disclosed. In one embodiment of the
invention, the vector comprises a plasmid. Moreover, host vector
systems for the production of a recombinant hematopoietic molecule of
the present invention are provided which comprise a vector of the
present invention in a suitable host, preferably a mammalian cell such
as a CHO or COS cell. This host vector system may be grown under
suitable conditions which permit the expression of the recombinant
lo hematopoietic molecule, which may be recovered by purification
techniques known in the art, e.g. ion exchange chromatography,
affinity chromatography, and size exclusion chromatography.
The present invention further provides pharmaceutical compositions
useful for treating patients suffering from anemias of various
origins, e.g. renal failure, and AIDS. Moreover, these pharmaceutical
compositions are useful for administering to patients for preoperative
autologous blood donations, patients rece-,ving or donating bone marrow
for transplantation purposes, and patients undergoing cancer
chemotherapy. These pharmaceutical compositions comprise effective
hematopoiesis-promoting amounts of a recombinant molecule of the
present invention and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are known in the art and are
d1sclosed in The Pharmacopeia of the United States and the National
Formulary. Depending on the speclfic application contemplated, the
pharm ceutlcal compositlon may be formulated as a solution,
suspenston, parenteral preparation, or spray. Parenteral preparations
may include a vehicle such as specially distilled, pyrogen-free water,
phosphate buffer, or normal saline. Oral and/or transmucosal dosage
forms may comprise phospholip~ds, often in the form of liposomes.
Also provided is a method for treating a patient to promote
hematopo~esis which comprises administering to the patient an
effective hematopoiesis-promoting amount of a pharmaceutical
composition of the present invention.
.. .. . . . ......... . ......... . . . ............... .
. . . . ..
WO 92/06116 ` . . ~ PCI`/US91/07053
Z~6-9746 ~
-- 10 --
The recombinant hematopoietic molecules, nucleic acid molecules,
pharmaceutical compositions and methods of the present invention will
be better understood by reference to the following experiments and
examples, which are provided for purposes of illustration and are not
to be construed as in any way limiting the scope of the invention,
which is defined by the claims appended hereto.
Examples
lo Construction of the hvbrid protein aenes: Genes encoding IL-3 (SEQ ID
NO: 16), Epo (SEQ ID NO: I7) and G-CSF (SEQ ID NO: I8) were purchased
from British Biotech. Ltd. These genes were utilized to construct
three different hybrid hematopoietic proteins, i.e., IL-3:Epo,
Epo:lL-3 and IL-3:G-CSF. In these hybrids the first named gene forms
the amino portion and the second named gene the carboxyl portion of
the hybrid protein.
ExamDle I
A nucleic acid molecule encoding an IL-3:Epo hybrid growth factor was
constructed as follows: CSF, the native leader sequence of IL-3 was
synthesized as 4 oligonucleotides (SEQ ID NOS: I9-22; see Table I)
which represents both strands of the leader sequence. In addition,
the 5' end of the leader (SEQ ID NO: I9) encoded a conven~ent
r~str1ctlon enzyme overhang (EcoRI), although the EcoRI site was not
regenerated, ~n front of the ATG start codon. The 3' end of the
leader (SEQ ID NO: 2I) included the first several amino acid codons of
IL-3 and an Spel overhang so that the annealed leader sequence could
be easily liSated to IL-3, which was altered by British Biotech to
~nclude an Spel slte. The leader sequence was annealed and l~gated to
pKS (Stratagene Cloning Systems, Inc., San Diego, CA) cleaved with
EcoRI and Spel. The resulting plasmid was destgnated pKSO. The IL-3
conta1ning pUC18 plasmid obtained from British Biotech was cleaved
with Spel and NheI, then l~gated to a linker oligonucleotide
3S (complimentary oligonucleotide SEQ ID NOS: 23 and 24; see Table 1)
which contained the following three restriction sites: Nhel, Xbal and
WO 92/06116 ,~ ~_, . PCI/US91/07053
Z~69~6i
-- 11 --
Ncol. Cleavage was then performed with SpeI and Xbal. The resulting
379 base pair fragment was then ligated to PKSO cleaved with
Spel and Xbal. The resulting plasmid (pKSOlL-a) contained the ll-3
leader, the IL-3 gene and a small linker fragment.
The Epo gene was inserted into pEe6 (Celltech, Ltd., Slough, U.K.), a
mam~alian expression vector which contains the human Cytomegalovirus
promoter, a polylinker region and a poly-A addition site in addition
to ampicillin resistance and a bacterial origin of replication, by
cleaving the Epo containing plasmid obtained from British Biotech with
Hindlll and BamHl. Epo was then cleaved with Ncol. The same linker
comprising oligonucleotide SEQ ID NOS: 23 and 24 as described earlier
was ligated to Epo and then cleaved with Xbal to yield the entire Epo
gene. This was then ligated to Xbal and BclI cleaved pEe6 to yield
pEe6 containing the Epo gene (pEepo). PKSOlL-a was cleaved with EcoRV
and an XbaI linker was ligated to the blunt ends followed by cleavage
with XbaI, which released the IL-3 gene with the leader sequence.
This was then ligated to Xbal cleaved Peepo to yield a plasmid
containing an entire hybrid protein gene (pEepie-a) (see SEQ ID NO: 11
for the structure of the inserted hybrid gene, designated herein IL-
3:Epo ShortJ. The glutamine synthetase (gs) gene was then inserted
1nto the 8amH1 site of pEepie-a to yield pEepogs-a or pEpogs-b,
depend1ng upon the orientation of the gs gene. Glutamine synthetase
confers res1stance to meth10n1ne sulphox1m1ne (MSX) in order to select
cells wh1ch have taken up the plasmid after transfection. After the
plasm1d was constructed a large batch was grown, purified by CsCl
ultracentrifugation, and used for transfection. At each step in this
process all ligat10n jo1nts between fragments were analyzed by DNA
sequence analysis in order to assure that there were no changes that
would cause framesh1fts and prevent the hybrid gene from being
expressed.
To construct a nucleic ac1d molecule encoding an IL-3:Epo hybrid
growth factor w1th a longer linker sequence separat1ng IL-3 and Epo,
pEep1e-a was cleaved with Nhel and annealed oligonucleotide SEQ ID
,,, . .. , . ~ , . .. . ...
, .... ~ . . .. . . ..
. . . . . . .. .
~ . ,, : . . : .. ..
' ! .. ' : ` ' ' ~ ' " ' ' ~ ~
WO 92/06116 PCr/US91/07053
2C'~97~6 ' ` ~
-- 12 --
NOS: 25 and 26 (see Table 1) were ligated into the c1eaved plasmid.
This linker encodes the flexible amino acid sequence Gly Ser Gly Ser
Gly Ser (SEQ ID NO: 27). Clones with the insert in the proper
orientation were selected by probing colonies with the junction
ollgonucleotide SEq ID NO: 28 (see SEQ ID NO: 14 for the structure of
the inserted hybrid gene, designated herein IL-3:Epo Flex). The
glutamine synthetase gene was then added to the construct as described
above.
1o ~ 1e 2
A nucleic acid molecule encoding an IL-3:G-CSF hybrid growth factor
was constructed as follows: pUC18 containing G-CSF (British Biotech)
was cleaved with Hind~II. A linker composed of an overhanging Xbal
site, a Notl site and an overhanging Hindlll site (oligonucleotide SEQ
ID NOS: 29 and 30; see Table 1) was ligated to the pUC18:G-CSF. This
was then cleaved with XbaI and BamHI which released the entire G-CSF
gene. The G-CSF fragment was then inserted into Xbal and BclI cleaved
pEe6 (pEe6:G-CSF). IL-3 with its signal sequence was removed from
the IL-3:Epo plasmid pEepogs-a as an Xbal fragment. This IL-3
fragment was then inserted into Xbal cleaved pEe6-G-CSF. After
restriction analysis, a plasmid containing the IL-3 gene in the proper
orientation was obtained (pEGII), this plasmid encoded a gene capable
of expresslng IL-3 and G-CSF as a hybrid protein (see SEQ ID NO: I3
for the structure of the inserted hybrid gene, designated herein IL-
3:G~CSF). The gs gene was inserted into th1s plasmid as described in
Example I above to yield plasmids pEGI3 and pEGI4, depending upon the
orlentatlon of the gs gene.
Exam~le 3
A nucleic acid molecule encoding an Epo:IL-3 hybrid growth factor was
constructed by first synthesizing the native Epo signal sequence as
oligonucleotide SEQ ID NOS: 31-36 (see Table 1). These were annealed
to yield an overhanging 5' Xhol sequence and a 3' Pstl sequence.
These were then ligated and subcloned as an Xhol/Pstl fragment
, .. ~ , , . . ~ . ,, ....... - ....... . .. . - -. . ..
', . . , ' , . , ., . . ~, .. .. . .. . . . .. . . .. . . .
w o 92/06116 Z~i9~46 ~ ~- . `` Pc~r/~S91/07053
- 13 -
(pEpol). In order to obtain the proper reading frame and signal
sequence processing site, the plasmid containing the signal sequence
was cleaved with PstI and the 3' overhang left by Pstl was
enzymatically removed with T4 polymerase. This was then cleaved with
BamHl. The Epo gene was then amplified by PCR as a fragment with a 5'
blunt end using oligonucleotide SEQ ID NO: 37 as a primer and a 3'
BamHl end using oligonucleotide SEQ lD NO: 38 as a primer. This
fragment was then ligated into pEpol to yield a complete Epo gene with
its leader sequence. PCR was used to amplify the Epo gene with its
lo signal sequence as an (5') Xbal and (3') Notl fragment using
oligonucleotide SEQ lD NOS: 39 and 40 as primers. This was then
digested with Xbal and Notl. At the same time, a purified IL-3
fragment was amplified by PCR as a (5'` Notl and (3') BamHl fragment
using oligonuc,eotide SEQ ID NOS: 41 and 42, followea by digestion
with Notl and BamHI. Ihese two fragments were ligated to pEe6 cleaved
with Xbal and Bcll to yield a full length hybrid gene encoding both
Epo and IL-3 (pEGI6) (see SEQ ID NO: l2 for the structure of the
inserted hybrid gene, designated herein Epo:lL-3 Short). Ihe gs gene
was inserted as described in Example l above to yield pEGl7 and pEGl8,
depending upon the orientation of the gs gene.
A flexible linker is inserted 1nto Epo:lL-3 by cleaving pEGl7 or pEG18
with Notl. Annealed oltgonucleotide SEQ ID NOS: 43 and 44 are then
11gated 1nto the cleaved plasm1d. Clones with the insert 1n the
proper ortentatton are selected by probing colonies with a junction
ollgonucleotlde as descr1bed above (see SEQ ID NO: l5 for the
structure of the 1nserted hybr1d gene.)
. .
- . ~ ,.. , , ... ; .. .... . . .
... -. ~ ~ . .; , . . ..
. ,-. : . . .. ... . .
. , ., . . .. . .,~ . . . -
. .; . , . . , . -
, . . ., - :
-. . . . . . .. .. .
WO 92/061 16 ; ` , . . PCr/US91/07053
- 14 -
Z C~Çi9~74L6 TABLE I
OLIGONUCLEOTIDES
All oligonucleotides are listed in the 5' to 3' orientation:
AATTGCCGCC ACCATGAGCC GCCTGCCCGT CCTGCTCCT (SEQ ID NO: 19)
GCTCC M CTC CTGGTCCGCC CCGGACTCCA AGCTCCCATG ACCCAGACAA (SEQ ID NO: 20)
CTAGTTGTCT GGGTCATGGG AGCTTGGAGT CCGGGGCGG (SEQ ID NO: 21)
ACCAGGAGTT GGAGCAGGAG CAGGACGGGC AGGCGGCTCAT GGTGGCGGC (SEQ ID NO: 22)
CTAGCGATCT TTCTAGA (SEQ ID NO: 23)
CATGTCTAGA MGATCG (SEQ ID NO: 24)
CTAG M GCGG CCGCA (.~EQ ID NO: 29)
TTCGCCGGCG TTCGA (SEQ ID NO: 30)
TCGAGCCATG GGGGTGCACG AATGTCCT (SEQ ID NO: 31)
GCCTGGCTGT GGCTTCTCCT GTCCCTGCTG TC (SEQ ID NO: 32)
GCTCCCTCTG GGCCTCCCAG TCCTGGGCTG CA (SEQ ID NO: 33)
GCCCAGGACT.GGGAGGCCCA GAGGGA (SEQ ID NO: 34)
GCGACAGCAG GGACAGGAGA AGCCACAGCC AGGCAGGACA TT (SEQ ID NO: 35)
CGTGCACCCC CATGGC (SEQ ID NO: 36)
GCCCCACCAC GCCTCATCTG T (SEQ ID NO: 37)
G M TTCGGAT CCTTATCATC T (SEQ ID NO: 38)
CTAGTCTCTA GMTGGGGGT CCACGAATGT (SEQ ID NO: 39)
AGCCATGGCG GCCGCTCTGT CCCCTGTCCT (SEQ ID NO: 40)
GACAGAGCGG CCGCCATGGC TCCCATGACC (SEQ ID NO: 41)
6 M TTCGGAT CCTTACTAAA AGATCGCTAG (SEQ ID NO: 42)
2S CTAGCGTCCG GAGGCGGTGG CTCGGGCGGT GGCGGCTCGG GTGGCGGCG GCTCTGCG
(SEQ ID NO: 25)
CTAGCGCAGA GCCGCCGCCA CCGCAGCCGC CACCGCCCGA GCCACCGCC TCCGGACG
(SEQ ID NO: 26)
TTGTCGCTAG CGTCCGGAGG C (SEQ ID NO: 28)
GGCCGCTTCC GGAGGCGGTG GCTCGGGCGG TGGCGGCTCG GGTGGCGGC GGCTCTGC
(SEQ ID NO: 43)
GGCCGCAGAG CCGCCGCCAC CCGAGCCGCC ACCGCCCGAG CCACCGCCT CCGGCAGC
(SEQ ID NO: 44)
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Example 4
Transfection of the hvbrid qene containinq Dlasmids. All
transfections were performed using the Lipofectinç transfection kit
S (Bethesda Research Labs, Gaithersburg, MD) using IS-30 mg. of purified
plasmid DNA (pEepogs-a, pEepogs-b, pEGI3, pEGI4, pEGI7, and pEGl8).
The following alterations were made to the protocol provided by the
company: the growth medium in these experiments was GMEM-S and the
CH0-KI cells were incubated in the presence of 10% C02; after addition
of the lipofectin:DNA complex, cells were incubated without selection
for 24 hours. The cells were transferred to GMEM-S supplemented with
25 mM MSX after 24 hours. The MSX concentration was subsequently
increased to S0 mM after one week. Cloning rings were used to
subclone MSX resistant colonies and each of these colonies was placed
into an individual well of a 24 well plate. Selected clones were
incubated in the absence of MSX to insure that the hybrid protein gene
was stably integrated. Strongly positive clones were grown in large
cultures to provide larger amounts of hybrid proteins for further
analysis.
_~am~le 5
Assavs for hvbrid protein production. Cell supernatants from
transfected or control cells were assayed using several different
assays. In order to demonstrate Epo production, an RIA kit for Epo
was used ~Incstar Corp., St111water, MN). The presence of IL-3 was
determ1ned uslng an ELISA assay 1n which the caoture antibody was a
polyclonal goat ant1-lL-3 (R&D Systems, Minneapolis, MN) and the probe
antlbody was a murine anti-lL-3 mor,oclonal. Goat anti-mouse
con~ugated to horseradish peroxidase followed by suitable substrate
was used to detect the presence of the monoclonal anti-lL-3. A very
s1m11ar assay was used to demonstrate the presence of the hybrid
proteins except that a murine ant1-Epo monoclonal or anti-6-CSF
monoclonal was used in place of anti-lL-3 monoclonal. Addit10nally.
IL-3:Epo Short was analyzed by Western blot analysis. The blo~ was
probed w1th antibody to Epo and then with I25I goat anti-mouse. A
. ' ' ' ` ' , ' ' ' ' ` `
w o 92/06116 26~9~7~6 PCT/~Sgl/070~3
- 16 -
single broad band appeared on the autoradiogram with a molecular
weight of slightly more than SO,OOO daltons.
ExamDle 6
Cellular assavs. Epo and/or IL-3 dependent and responsive cell lines
were used to test the biological activities of the hybrid proteins.
B6SUtA (5) is a multipotential hematopoietic progenitor cell line
established from nonadherent cell populations removed from continuous
e6.s mouse bone marrow culture. ~his cell line demonstrates absolute
dependence upon a source of growth factor(s). In response to Epo a
population of the cells synthesize hemoglobin. Studies of globin
expression indicated that the globin programs of B6SUtA ce,ls are
similar to those of erythroid progenitors at the period of transition
from the yolk sac to fetal liver erythropoiesis. TF-I (6) it is a
cell line of immature erythroid origin established from a patient with
erythroleukemia. The cell line shows complete dependency on GM-CSF or
IL-3. Epo sustains short-term growth of TF-I and will induce
hemoglobin synthesis in a very small population of cells (870). Hemin
and w-aminolevulinic acid induce hemoglobin synthesis in most of the
cells.
Human IL-3 wlll not bind the murine IL-3 receptor, therefore
experiments that were done with B6SUtA cells measured only the
funct10nallty of the Epo molety of the hybrid. B6SUtA cells are
carr1ed 1n murine IL-3. In each experiment, they are washed
thoroughly and set up wtth growth factors at 105 cells/ml. Cell
growth and hemoglobin content were monitored on days 3 and 6 of each
experiment. Cells grown in the presence of concentrated (IOX) CHO
condltioned medium (CM) conta1ning IL-3:Epo Short at a final
concentrat10n equivalent to 4.8 units/ml of Epo grew as well as cells
grown in an equivalent amount of recombinant human (rHu) Epo. The
percentage of cells which synthesized hemoglobin in response to the
CHO-lL-3:Epo Short CM was always four times that of cells exposed to
~S rHu Epo. B6SUtA cells grown in the presence of rHu IL-3 and rHu Epo
grew as well as cells grown in the presence of IL-3:Epo Short and
.. . . . . .
.
' .
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induced hemoglobin synthesis in the sa~e percentage of cells as did
rHu Epo. Cells exposed to recombinant murine IL-3 (rMu IL-3) and rHu
Epo grew similarly to cells exposed to rMu IL-3 alone and neither
effectively induced cells to synthesize hemoglobin. Concentrated
control CHO CM did not support the growth of B6SUtA cells nor did it
induce hemoglobin synthesis. CHO CM plus rHu Epo supported cell
growth and hemoglobinization as well as CHO-lL-3:Epo Short CM.
CHO-lL-3:Epo Short CM as well as CHO-rHu IL-3 CM both supported growth
lo of human TF-I cells. Control CHO CM supported only limited growth of
the TF-I cells.
Oiscussion
The above-mentioned results demonstrate that a hybrid protein
comprising two growth factors can be expressed in mammalian cell
culture systems. ln vitro assays of IL-3:Epo Short indicate that this
hybrid protein has the activities of both IL-3 and Epo. The
therapeutic application of such hybrid factors has advantages over
using two factors separately simply in terms of patient
administration, and moreover since the production, purification and
formulation of one factor is less labor intensive than for two
separate factors.
2S Exaoplo 7
Fac~or ~ependent Cell Lines and Culture Media - The GM-CSF/lL-3/Epo
dependent human TF-I cell line and the G-CSF dependent murine NFS-60
cell line were grown and maintained as described (7,8,). The GM-CSF
dependent human cell 11ne AML 193 (9) was adapted for growth ln IL-3
by cont1nuous cultu~e of the cells in RPMI-1640 plus 10% FCS
supplemented with rHu IL-3 for 6 weeks. The TF-l derived cell line,
TF-136 was selected by continuous culture of the TF-I line in
RPMI-1640 plus lOX FCS supplemented with 5ng/ml of rHu IL-3 for 6
3S months, followed by single cell suspension cloning of the resultant
IL-3 dependent cells. The Epo dependent murine cell line, FDC-PI/ER,
. - . - . . . .
". ,. . :. . ,......... . . ,. ., . : , . -
.. :......... . . . :,, . : - .
: - . - . .:, ~, ; ... . ..
: . . , . . ,., . , . . .:- ~ .
Z~9~46
WO92/06116 . ~ . . PCI/US91/07053
,.``' ~
- 18 -
was derived from the IL-3 dependent line, FDC-PI, by introduction of
the murine Epo receptor into these cells. (10) FDC-PI/ER cells are
maintained in RPMI-1640 plus 10% FCS supplemented with I unit/ml of
rHu Epo. Recombinant human Epo was obtained from Ortho Biologicals,
S Inc (Raritan, NJ). Recombinant human IL-3, rHu G-CSF and rHu GM-CSF
were purchased from R ~ D Systems (Minneapolis. MN).
Capture ELISA Assay - ELISA plate was coated with 5 ~9/200 ~l/well of
goat anti-human IL-3 (R & D Systems) in PBS at 40C overnight. Excess
lo antibody was remov~d by washing with PBS. Blocking was carried out
with 300 ~l/well of 1% non-fat milk in PBS for I hour at 37C followed
by washing with 0.05% Tween~ in PBS. Samples were then incubated with
the IL-3 antibody for 1 hour at 37DC in 0.5% non-fat milk, 0.02s%
Tween~. Following extensive washing, the second antibody, a mouse
anti-Epo monoclonal (Genzyme, Cambridge, MA ), was added to the plate
which was incubated for I hour at 37~C. The plate was washed and
incubated with conjugate antibody (Goat anti-mouse-horseradish
peroxidase) for 30 minutes at 37C. Color development was carried out
with the addition of o-phenylenediamine/H202 at room temperature (RT)
for 30 minutes. The reaction was stopped with lN H2S04 and the
samples were read at 495 nm.
Gene A~pliftcatton- CHO cell lines productng signiftcant amounts of
the hybrld growth factors were lsolated and 106 cells were plated tn
a 75 mm T-flask ln GMEM-S medtum containtng various concentrations of
methlonlne sulphoxlmlne (MSX), ranging between lOO~M and SOO~M.
Colontes reststant to the h1ghest MSX concentratton (IL-3:Epo Flex
200~M: IL-3:Epo Short 250~M; Epo:lL-3 Short 250~M; IL-3:G-CSF 250~M)
were tsolated and expanded. Those clones productng the htghest levels
of hybrtd growth factors as determined by Epo or G-CSF ELISA assay
(Amgen) were used for subsequent studtes. IL-3:Epo Flex (clone
23-10); IL-3:Epo Short (clone 5-4); Epo:lL-3 Short (17-3-1).
Cell Prol1ferat10n Assays - Factor dependent cells were grown to
3S st-t10nary phase, washed, and lncubated for 16 hours tn media plus
IOX FCS deprived of growth factor. The cells were plated at a
WO 92/06116 2~9~46 PCI`/US91/07053
-- 19 -- ~.~
concentration of 2 X 105 cells/ml in a 96 well microtiter plate (200
~l/well) with and without growth factor. Recombinant human growth
factors were diluted into CH0 conditioned medium (CM) before addition
to cells. Following incubation for 42 hours~ (3H) thymidine (I ~Ci/
well; New England Nuclear, Boston. MA) was added and the cells were
incubated for another 6 hours. The cells were then hypotonically
lysed and harvested onto glass fiber filters. The filters were washed
with distilled water, dried and counted in liquid scintillation fluid.
Bone Marrow Cultures - Informed consent was obtained prior to
aspirating bone marrow from normal volunteers. Aspirated bone marrow
was diluted I:I in ~- medium without nucleosides containing
preservative-free sodium heparin. A single cell suspension was
prepared, layered, over an equal volume of Ficoll-Hypaque (sp gr 1.077
g/ml) and then centrifuged for 25 minutes at 1,500 rpm at 40~C. The
light-density mononuclear cells were collected and washed and diluted
to S XI05 cells/ml with Iscove's modified Dulbecco's medium plus 20%
modified FCS (Gibco BRL). Cells (1.25 X I05 /ml) were plated in 0.8X
methylcellulose supplemented with various concentrations of rHu Epo,
rHu IL:3 and hybrid growth factors. Cultures were incubated for
either 7 or I4 days in a humidified atmosphere with 5% C02 at 37~C.
Colonies were counted at day 7 for CFU-E and at day I4 for BFU-E under
an 1nverted mlcroscope.
Western Blot Analysis - CH0 CM containing approximately 75 ng of IL-
3:Epo fuslon prote1n was electrophoresed on a 10-20% gradlent SDS PAGE
gel (Integrated Separations Systems) under reducing and denaturing
cond1tions. Samples were loaded in 0.0625 H Tris-HCl (pH 6.8), 2X
SDS, 5% 2~mercaptoethanol, IOX glycerol and -0.002% bromophenolblue
follow~ng heat treatment at IOO-C for 3 minutes. The proteins were
transferred to nitrocellulose (Bio Rad) in 25 mM Tris, I29 mM glyc1ne,
pH 8.3, 20% methanol, at 150 V, constant power, for 90 minutes. The
transfer efficiency was monitored by visual examinatlon of the
completeness of transfer of prestained molecular weight markers (Bio
Rad). The nitrocellulose membrane was incubated in PBS containing 3X
BSA for I hour at room temperature and subsequently washed in PBS
~ . . ..
.
., ,, , .~ ~ . ' , . ' ' ! ' . . ' .
WO 92/06116 . PCI`/US91/07053
?
2 ~ 3~ 20 -
containing 0.5% Tween (PBS-T) for 5 minutes at room temperature. The
membrane was probed with primary anti-Epo anti-sera in 3% BSA in PBS.
Excess antibody was removed by 3, 5 minute room temperature washes in
PBS-T. The nitrocellulose membrane was then probed with a secondary
antibody conjugate (Goat anti-Rabbit IgG/ Alkaline Phosphatase, Bio
Rad) for I hour at room temperature. Excess secondary antibody was
removed by two washes with PBS-T as above. Color development was
carried out by incubation with color reagents (Bio Rad) in alkaline
phosphatase buffer (100 mM Tris HCl, pH 9.5, 100 mM NaCl, S mM MgC12).
The reaction was stopped by immersion of the membrane in cold (4C)
distilled H20.
Results and Discussion
HY~rid arowth factor Dlasmid amDlification. Individual transfected
CH0 cell c-10nes producing significant amounts of the desired hybrid
growth factor were identified by ELISA capture assay, Table Il. The
clones were plated out and placed in medium with increasing
concentrations of MSX, ranging between 100 ~M and 500 ~M. Colonies
survlving at the highest concentration of MSX were isolated and grown
to confluence. Serum and drug-free medium was then added to the
cells and collected after 4 days. At the time of collection fresh
serum and drug-free medium was added to the cells. A total of 3
collections were taken. The amount of hybrid growth factor produced
ln the collectlons was determined by Epo or G-CSF ELISA assay (Table
III) and appropriate collections were pooled. ~he pooled CM was used
as a source of hybrid growth factors ~n all cellular assays.
.. ~ ., ...... ... , . .. . , ., ,. ,. .~ . ~ .. ... ,.. ,-...... . ....... .
, - - i: , , : -
:, . .. . , ,.,. . :. .. , . . ,- . -, ,. -, . - , ... . . : ..
w o 92/06116 PCT/~S91/07053
~ 2~97~6 ~.
- 21 -
TABLE II
HY~rid Growth Factor OD (495 nm)
CHO CM* 0.24
IL-3:Epo Flex 2.44
IL-3:Epo Short 2.46
Epo:IL-3 Short 1.59
IL-3:G-CSF 2.40
* Conditioned medium from CHO cells transfected with the vector
pEe6.
TA~LE III
Hybrid Growth Factor CollectionConcentration (~a/ml)*
IL-3:Epo Flex 1st 3.0
2nd 4.2
3rd 3.3
IL-3:Epo Short 1st l.S
2nd 5.8
3rd 6.7
Epo:lL-3 Short Ist 26.7
2nd 53.3
3rd 58.7
IL 3:G-CSF Ist 2.2
2nd 2.0
3rd 2.0
~ Concentrations were determined by Epo and G-CSF ELISA Assay.
Detectlon of hvbrid growth factor Droduction. In order to confirm
that the IL-3 and Epo detected in the ELISA capture assays were belng
produced ln the form of a fusion protein, ~estern blot analysis was
performed. Cond1tloned medium from CHO cells transfected with IL-
3:Epo Flex cONA (Figure 1, lane 2), IL-3:Epo Short cONA (Figure 1,
lane 3) and Epo:lL-3 Short cDNA (Figure 1, lane 4) were probed with
3S mouse ant~-Epo polyclonal anti-sera. Immunoreactive material
corresponding to a molecular weight of approximately 50,000 daltons.
.- . . ~ ; .. - . .. . - . .. . ;-....... ....
WO 92/06116 PCr/lJS91/07053
Z~i9746 ~ ~
-- 22 --
the expected size of the IL-3:Epo and Epo:IL-3 hybrid growth factors,
was detected in each sample. Comparison with increasing
concentrations of rHu Epo (Figure I, lanes 6-I0) indicated that the
antibody used in this analysis recognizes the Epo moiety of the fusion
proteins efficiently.
IL-3 bioactivitY of the IL-3:EDo and Epo:lL-3 hYbrid qrowth factors
To determine whether the IL-3 moiety of the IL-3:Epo and Epo:IL-3
hybrid growth factors was functional, its ability to support growth of
the IL-3-dependent human cell line, AMLI93, was evaluated (Figure 2).
As Epo does not s~pport growth of these cells (Figure 2), only IL-3
activity was measured in this assay system. CH0 CM containing rHu IL-
3 and levels of hybrid growth factors sufficient to support maximal
proliferation were added to the culture medium. The cells were then
pulsed with (3N) thymidine and the radioactivity incorporated into the
DNA was used as a measure of cell growth. Cells exposed to CH0 CM
containing no growth factors, supported the proliferation of AMLI93
cells to the same extent as did cells grown in medium alone. Each of
the fusion proteins when present in excess, supported the growth of
AMLI93 cells in a manner equivalent to that of rHu IL-3.
The functional activity of the IL-3 portion of the IL-3:Epo and
Epo:lL-3 hybrid growth factors was further evaluated by comparing the
fusion proteins to rHu IL-3 in dose response experiments (Flgure 3).
2S The incorpor~tion of (3H) thymldlne into AMLI93 DNA was again used as
mN~sure of cell prollferatlon. When IL-3 was located at the N-
termlnus of the hybrid growth factor prote1n (IL-3:EpoJ, its ability
to support AMLI93 proliferation was equivalent to that of rHu IL-3
~D50 ~ 5 fmol/ml). Size (2 aa versus 23 aa) and flexibility of the
linker did not greatly impact the function of the IL-3 moiety.
However, when IL-3 was located at the C-terminus of the fusion Drotein
(Epo:lL-3), its ability to support the growth of AMLI93 cells was less
(ED50 ~ 200 fmol/ml) than that of rHu IL-3 and the IL-3:Epo hybrid
factors. These results suggest that linkage of IL-3 at the N-terminus
interferes with function while linkage at the C-terminus does not. It
h~s prev1Ously been reported that modification of the C-terminus of
-, ~ " "
w o 92/06116 ;~C~j9'746 pcT/~s9l/o7n~3
'
- 23 -
murine IL-3 did not interfere with its activity (11). Therefore, it
should be possible to target any molecule or compound of interest to
cells expressing the IL-3 receptor through linkage to IL-3 at its C-
terminus.
ED. bioactivitY of the IL-3:EDo and Epo:IL-3 hvbrid qrowth factors.
To determine whether the Epo moiety of the hybrid growth factors was
functional, its ability to support the growth of the Epo-dependent
murine cell line FDC-PI/ER, was evaluated (Figure 4). This line
o derived from FDC-PI cells expresses t~;e murine Epo receptor (I0), and
is dependent on either murine IL-3 or Epo (murine and human) for
growth (Figure 4). As IL-3 is a species specific growth factor,
murine IL-3-deDendent cells do not respond to human IL-3 (I2).
Therefore, when using the FDC-PI/ER cell line to evaluate
functionality, only the activity of the Epo moiety is measured. CH0
CM containing rHu Epo and levels of hybrid gro~th factors sufficient
to support maximal proliferation were added to the culture medium.
The cells were then pulsed with (3H) thymidine and the radioactivity
incorporated into the DNA was used as a measure of cell growth. Cells
exposed to CH0 CM which did not contain cytokines did not support the
proliferation of FDC-PI/ER cells. Each of the fusion proteins when
present in excess, stimulated the growth of FDC-PI/ER cells to the
same extent as did rHu Epo. (Flgure 4)
The blologlcal function of the Epo portlon of the IL-3:Epo and Epo:lL-
3 hybrld growth factors was further analyzed by comparing the fusion
protelns to rHu Epo in dose response experlments (Figure 5). The
lncorporation of (3H) thymidine into FDC-PI/ER cells was used as a
measure of cell proliferation. Each of the hybrid growth factors was
equlvalent to rHu Epo in ability to stimulate prollferatlon of FDC-
PI/ER cells ~ED50 ~ 50 fmol/ml) . Slze (2-3 aa versus 23 aa) and
flexlb111ty of the llnker, as well as the orientation of Epo within
the protein (N-terminus versus C-terminus) did not alter function.
Evidence exists suggesting that the N-terminus of Epo is not involved
3S in receptor bindlng as a monoclonal antibody directed toward the N-
terminus of Epo does not neutralize its activity (13). The results
.. . ~ .. . . . . ~. . ............... .. . . ... .~ - -
. . .. . . ... . . . ... . . . . . .
.: . . - . :- . ... - -, - .
WO 92/06116 PCI'/US91/070~3
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-- 24 --
presented here suggest that linkage of Epo to a second protein does
not impair its abi1ity to bind its receptor or transduce a signal.
Epo could therefore be useful as a carrier protein which would target
a molecule or compound of interest to those cells expressing the Epo
receptor.
IL-3 ~lus EDO bioactivitv of the IL-3:Epo and Epo:lL-3 hvbrid arowth
factors. In order to study the effects of IL-3 and Epo in
combination, proliferation of a human cell line, TF-I (7), dependent
on IL-3 and Epo for growth was measured. This experiment was done on
a cytokine weight basis and the results are represented on a molar
basis (Figure 6). rHu IL-3 (R & D Systems) made in E. coli is
nonglycosylated. rHu Epo and hybrid growth factors made in CH0 cells
are glycosylated. Therefore, when equal weights of the growth factors
were added to the cell culture medium, approximately twice the number
of unglycosylated molecules of IL-3 were added as compared to
glycosylated Epo and hybrid growth factor molecules.
CH0 CM containing rhu IL-3 plus rHu Epo and levels of hybrid growth
factors which support suboptimal proliferation of ~F-I cells adapted
for growth in IL-3 were added to the culture medium. Cell growth was
monitored by radioactivity incorporated tnto the DNA. (Figure 6) The
act1~ittes of IL-3 plus Epo were not synergistic in this cell ltne,
nor were they addlttve. At these low levels, the activities of the
2S IL-3:Epo Flex and IL-3:Epo Short fuston protetns were comparable to
those of ~ m1xture of the two cytoklnes. Epo:lL-3 Short activity was
aga1n reduced in compartson to that of the IL-3:Epo hybrid growth
factors and the combination of IL-3 plus Epo. This is likely to be
due to decreased IL-3 acttvity.
The btologtcal acttvity of the IL-3:Epo and Epo:IL-3 hybrid growth
factors was further evaluated in dose response experiments (Figures 7
~ 8). TF-I cells adapted for opttmal growth in IL-3 were exposed to
CH0 CM contatning hybrid growth factors (Figure 7). Each of the
fuston prote~ns when present in excess were able to support growth of
the cells to the same extent (Figure 7A). At lower doses, the IL-
'' ' '~.', ' ` ' `' :.' ,", ' ',.. ., '"'' ',',' '.'', ' '' '.' ;.,'' ' '
WO92/06116 PCI/US91~07053
Z~i9746
-- 25 --
3:Epo Flex protein appeared to be slightly more potent than the shortlinkered hybrids (IL-3:Epo Flex ED50 = 0.37 fmol/ml; IL-3:Epo Short
EDSO ~ 0.75 fmol/ml; Epo:ll-3 Short ED50 = 0.9 fmol/ml) (Figure 7B).
Thls result was more pronounced in experiments done with TF-l cells
adapted for optimal growth in GM-CSF (Figure 8). The IL-3:Epo Flex
protein was dramatically more potent than the hybrid growth factors
containing short linkers (IL-3:Epo Flex ED50 = 0.07 fmol/~l; IL-3:Epo
Short and Epo:IL-3 Short ED50 = 0.75 fmol/ml (Figure 8B). When
present in excess with the GM-CSF adapted TF-l cells, each of the
fusion proteins stimulated cell proliferation to a similar extent
(Figure 8A). These results suggest that when IL-3 and Epo are fused,
the 23 aa flexible linker allows more efficient recep~or interaction
than does a short (2-3 aa) linker.
It appears that induction of receptor expression is possible by
growing a cell in the presence of a cytokine whose receptor it has the
potential to express. An up regulation of Epo receptor expression has
been reported in IL-3-dependent cells transferred into mediu~
supplemented with Epo (l4). Thus, it is likely that growing TF-1
cells in IL-3 or GM-CSF, preferentially increases the appearance of
cell surface IL-3 or GM-CSF receptors. Several research groups
(15-20) have observed a subset population of GM-CSF and IL-3 receptors
on prlmary human cells and hematopoietic cell lines capable of binding
both GM-CSF and IL-3. It has been suggested that a single accessory
2S molecule preferent1ally lnteracts wlth this subset of GM-CSF/IL-3
receptors allowing the transduction of signal. Our results raise the
posslb111ty that GM-CSF could be inducing the expression of an
accessory molecule in TF-I cells which may be important for binding
and could posslbly link IL-3:EPO signal transduction. This protein
could be identlcal to the GM-CSF/IL-3 receptor accessory prote1n.
~IYIl~lLig~ Dy formation stimulated bv IL-3:EDo and EDO:IL-3 hvbrid
growth factors, To assess the biological activity of the fusion
proteins on normal hematopoietic progenitor cells, analysis of the
3S formation of erythroid ~BFU-E and CFU-E) colonies fro~ nonadherent
mononuclear human bone marrow cells was performed. (Table IY) As was
. . . . ..
WO 92/061 16 PCT/US91/07053
~!
ZC~9~46 - 26 - .
observed with the cell lines, the Epo roiety of each of the fusion
proteins was equally active (CFU-E formation) on bone marrow
progenitor cells. The IL-3:Epo Flex protein was the most active
hybrid growth factor while the Epo:lL-3 Short protein was the least.
(It stimulated two-thirds the number of BFU-E as did the IL-3:Epo
Flex.) These results suggest that the IL-3:Epo Flex fusion protein
may have significant clinical benefits where indications for
combination therapy with IL-3 and Epo ~ay prove efficacious.
TABLE IV
Growth Factors BFU-Ea'b ~E~ ~c
No Factors
0.2 pmol/ml IL-3
O.I pmol/ml Epo + ~++
0.2 pmol/m~ IL-3 &
0.1 pmol/ml Epo +++ +++
O.l pmol/ml IL-3:Epo Flex+++ +++
O.I pmol/ml IL-3:Epo Short ++ +++
O.I pmol/ml Epo:lL-3 Short + +l+
a Mononuclear human bone marrow cells were used as a target cell
population.
b BFU-E were counted 14 days after plating. -
2S c CFU-E were counted 7 days after plating.
IL-3 b1Oactivitv of the IL-3:G-CSF hYbrid ~rowth factor. To determine
whether the IL-3 moiety of the IL-3:G-CSF hybrid growth factor was
functional, its abll1ty to support growth of the IL-3-dependent human
cell line TF-I, was evaluated in a dose response experiment (Figure
9). Quantitation of IL-3:G-CSF protein in CHO CM was performed using
a G-CSF ELISA assay in which the standard is unglycosylated G-CSF.
S1nce the IL-3:G-CSF fusion protein is glycosylated, measurements are
approxim te. G-CSF does not support growth of TF-I cells (Figure 9),
therefore, the only activity measured in this assay system was IL-3.
.. .~ .... ..... . . , .- - ~ - .- ; ........... .-
WO 92/06116 PCI`/US91/07053
2~ 7a~6 - 27 -
CH0 CM containing rhu IL-3, rHu G-CSF, and IL-3:G-CSF hybrid growth
factor were added to the culture medium. The radioactivity
incorporated into the DNA was used as a measure of cell proliferation.
CH0 CM did not support growth of TF-I cells. The mixture of rhu IL-3
plus rHu G-CSF stimulated proliferation to the same extent as did rhu
IL-3 alone. The IL-3:G-CSF hybrid growth factor induced a dose
response similar to that observed with IL-3.
G-CSF bioactivitv of the IL-3:G-CSF hvbrid qrowth factor. To evaiuate
the biological function of the G-CSF moiety of the IL-3:G-CSF hybrid
growth factor, its ability to stimulate proliferation of the murine
cell line, NSF-60, was tested. (Figure I0) G-CSF, unlike IL-3 is not
species specific, therefore, human G-CSF will actively cupport growth
of murine cells (2I). Cells exposed to CH0 CM containing no growth
factors, supported the proliferation of NSF-60 cells to the same
extent as did cells grown in medium alone. The IL-3:G-CSF hybrid
growth factor stimulated growth in a dose dependent manner equivalent
to that observed with G-CSF.
IL-3 ~ 1us G-CSF bioactivitv of the IL-3:G-CSF hvbrid arowth factor.
The biological function of the IL-3:G-CSF hybrid growth factor was
evaluated by its ability to support growth of an IL-3-,
G-CSF-dependent human cell line, AMLI93. rHO CM containing rHu IL-3,
rHu 6-CSF and IL-3:G-CSF hybrid growth factor were added to the
2S culture medlum. Cell proliferation was monitored by incorporation of
radloactlvlty lnto the DNA. (Figure II). Both IL-3 and G-CSF
supported growth of thls cell line ln a dose dependent manner. The
two cytokine activities were not synergistic. nor were they additive.
The IL-3:G-CSF hybrid growth factor stimulated AMLI93 proliferation to
a greater extent than did the mixture of the two cytokines.
. - . . . . , , . -. - .
- ~ ~ - . ~ , .. . ... ; . . ... . . . ... . .
WO 92/06116 2~ PCl'/US91/07053
.. ~ ...
- 28 -
REFERENCES
1 Sonoda, et a1., Proc. Natl. Acad. Sci. USA. 85:4630-4364 (1988).
2. Migliaccio, et al., Blood, Vol. 72, No. 3, 844-851 (1988).
3. Sieff, et al., Blood, Vol.73, No. 3, 688-693 (1989).
4. Fraser, et al., Exp. Hematol., 16: 769-775 (1988). -~
5. Enver, et al, Proc.Nat~. Acad. Sci. USA, 85:9091-9095 (1988).
6. Kitamura, et al.. J. Physiol., 140:323-334 (1989).
7. Kitamura, et al., J. Cell. Physiol., 140 : 323-334 (1989).
8. Holmes, et al., Proc. Natl. Acad. Sci. USA, 82 : 6687-6691 ~ -
(1985).
9. Lange, et al., Blood, 70 : 192-199 (1987).
10. Carroll, et al., J. Biol. Chem., in press (1991).
11. Dunbar, et al., Science, 245 : 1493-1496 (1989).
2S
12. Yang, Qt al., Cell, 47 : 3-10 (1986).
13. Sue, et al., Proc. Natl. Acad. Sci. USA, 80 : 3651-3655 (1983).
li. ~akaguchi, et al., Biochem. Biophys. Res. Commun., 146 : 7-12
~ (1987).
; 15. Park, et al., J. Biol. Chem., 264 : 5420-5427 (1989).
~ 35 16. Gesner, et al., J. Cell. Phys~o!., 136 : 493-499 (1988).
~ .
~'~
.
WO 92/06116 PCT/US91/070~3
~ 2~69;?46
- 29 -
17. Elliot, et al., Blood, 74 : 2349-2359 (1989).
18. Lopez. et al., Proc. Natl. Acad. Sci. USA, 86 : 7022-7026
(1989).
19. Budel, et al., Blood, 75 : 1439-1445 (1990).
20. Cannistra, et al., J. Biol. C~em., 265 : 12656-12663 (1990).
21. Souza, et al., Science, 232 : 61-65 (1986).
22. Umemura et al., Proc. Natl. Acad. Sci. USA, 85 : 9278-9282
(1988).
lS 23. Udupa, et al., Blood, 53 : 1164-1171 (1979).
24. Papayannopoulou, et al., J. Clin. Invest., 51 : 1179-118
(1972).
25. Migliaccio, et al... Blood, 72 : 944-951 (1988).
WO 92/06116 PCI'/US91/07053
2~i9746 ~'
- 30 -
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iJ APPLICANT: Rosen, Jonathan 1.
(~i) TITLE OF INVENTION: HYBRID GROWTH FACTORS
(iii) NUMBER OF SEQUENCES: 44
(iv) CORRESPONDENCE ADI)RESS: ~.
(A) ADDRESSEE: R~bert L. Minier
(B) STREET: 1 Johnson & Johnson Plaza
(C) CITY: New Brunswick
(D) STATE: New Oersey
(E) COUNTRY: USA
(F) ZIP: 08933
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOfTWARE: Patentln Release ~1.0, Version ~1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: 435
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/589,958
(B) FILING DATE: 28-SEP-1990
(vtii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Stark, Mlchael
~B) RE61STRATION NUMBER: 32,495
~C) REFERENCE/DOCKET NUMBER: BCI-15
(lx) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-524-2817
(B) TELEFAX: 908-524-2808
(C) TELEX: 844-481
(2) INFORMATION FOR SEQ ID NO:1:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGIH: 133 amino acids
(B) TYPE: amino acid
(C) STRANOEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: prote~n
.
.
WO 92/06116 PCr/US91/07053
~ z~ 9746
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Ser Asn Met Ile ASD Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu
Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala
Val Lys Ser Leu Gln Asn Ala Ser Ala lle Glu Ser lle Leu Lys Asn
Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro
9S
Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr
100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu
115 120 l25
Ser Leu Ala Ile Phe
130
~2) INFORMATION FOR SEQ 1D NO:2:
(1) SEQUENCE CHARACTERISTICS:
A~ LENGTH: 79 amino acids
B) TYPE: am1no ac1d
C) STRANDEDNESS: single
(D) TOPOLOGY: 11near
(~i) MOLECULE TYPE: protein
(ii1) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
, . - . . ~ .......... ~ .. -. - . .. -
- . . - . - - - ......... .. .-- . . - . .-
. .... .. . .. -. ~ - . - .
WO 92~ ,"~ PCI'/US91/07053
-- 32 --
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 s 10 15
Ser Asn Met lle Asp Glu lle lle Thr His Leu Lys Gln Pro Pro Leu
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu
Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala
SO 55 60
Val Lys Ser Leu Gln Asn Ala Ser Ala lle Glu Ser Ile Leu Lys
65 70 75
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE-TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Pro Pro Arg Leu lle Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu
1 5 10 15
Leu Glu Ala Lys Glu Ala 61u Asn lle Thr Thr Gly Cys Ala Glu His
Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Yal Asn Phe
Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val TrD
Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp
9S
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
100 105 110
~` . ' - t ; , .......................... . . "
- ', ,., ~ , , ' ' ' . ' " , `, ,. ~ ' - ' ; ' '
.. . . .. . . . .
WO 92/06116 2~9~46 PCI/U~9-~07053
' , . '
-- 33 --
Gly Ala Gln Lys Glu Ala lle Ser Pro Pro Asp Ala Ala Ser Ala Ala
115 120 125
Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val
130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala
145 150 155 160
Cys Arg Thr Gly Asp Arg
.
(2) INFORMATION FOR SEQ ID NO:4:
(i) sEquENcE CHARACTERISTICS: -
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iil) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala
1 S 10 15
Glu Asn lle Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn
lle Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met
Glu Val Gly Gln Gln Ala Yal Glu Val Trp Gln Gly Leu Ala Leu Leu
Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln
Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu
9S
Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala
100 105 110
.. ~ . . . ~ , , . ., . . . ~ . . . ... . .. , - .
WO 92/06116 - PCT/US91/07053
: . ` - .~,. . .
2~69746
-- 34 --
Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr
115 120 125
Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg
130 135 140
Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys
145 150 lSS
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 ~mino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) ~OPOLO&Y: linear
(ii~ MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys
1 5 10 15
Cys Leu Glu Gln Val Arg Lys lle Gln Gly Asp Gly Ala Ala Leu Gln
Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys Hls Pro Glu Glu Leu Val
3S 40 45
L w Leu Gly Hls Ser Leu Gly lle Pro Trp Ala Pro Leu Ser Ser Cys
SO 55 60
Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser
Gly ~eu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly lle Ser
9S
Pro Glu Leu 61y Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp
100 lOS 110
Pho Ala Thr Thr lle Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
115 120 12S
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe
130 135 140
., . . , . . . . . . . - , .
. . - . . - . ~ -: :. - : . . -
.. . -- :, . .. .: . ... . : . . .. .- . .
WO 92/06116 2~!697~L6 PCI/US91/070~3
,:~
- 35 -
Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe
145 150 155 160
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170
~2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 302 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Ser Asn Met lle Asp Glu lle lle Thr His Leu Lys Gln Pro Pro Leu
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu
Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala
Val Lys Ser Leu Gln Asn Ala Ser Ala lle Glu Ser lle Leu Lys Asn
Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro
lle Hls lle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr
100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala lle Phe Leu Asp Met Ala Pro Pro Arg Leu lle Cys Asp
130 135 140
Ser Arg Val Leu Glu Arg Tyr Leu Leu G1u Ala Lys Glu Ala Glu Asn
145 150 155 160
. . .. : .- .... -. . . . ~: ..
WO 92/06116 ` - PCI/US91/07053
@~
Z¦~jg~L~ ~ 36
Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr
165 170 I75
Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val
180 I85 I90
Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu
195 200 205
Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro ~rp
210 215 220
Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser
225 230 235 240
Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser
- 245 250 255
Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp
260 265 270
Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys
275 280 285
Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg
290 295 300
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 321 amino ac1ds
(B) TYPE: amlno ac1d
C) STRANDEDNESS- s1ngle
D) TOPOLOGY: 11near
(ll) MOLECULE TYPE: prote1n
(111) HYPOTHETICAL: NO
(1v) ANTI-SENSE: NO
(x1) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
Ser Asn Met Ile Asp Glu lle Ile Thr His Leu Lys Gln Pro Pro Leu
`20 25 30
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu
. ; , . . : ~
. -
'
`, . ~ . ' ' ' `. ' .
WO 92~06116 ~ 9~46 ` . ` PCr/US91~07053
-- 37 --
Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala
Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser lle Leu Lys Asn
Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro
Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr
100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ser Gly Gly Gly Gly Ser Gly Gly 61y Gly Ser Gly Gly
130 135 140
Gly Gly Ser Ala Leu Ala Ile Phe Leu Asp Met Ala Pro Pro Arg Leu
145 150 lss 160
Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu
165 170 175
Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu
180 185 190
Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg
195 200 205
Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu
210 2l5 220
Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser
225 230 235 240
61n Pro Trp 61u Pro Leu 61n Leu Hls Val Asp Lys Ala Val Ser Gly
245 250 255
Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu
260 265 270
Ala lle Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile
275 280 285
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu
290 295 300
Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp
305 310 315 320
Arg
0 92/06~ 7 ~6 PCT/~S91/07053
- 38 -
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 303 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ala Pro Pro Arg Leu lle Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu
I S 10 15
Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His
? 25 30
Cys Ser Leu Asn Glu Asn lle Thr Val Pro Asp Thr Lys Val Asn Phe
Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp
Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp
9S
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
100 105 110
Gly Ala Gln Lys Glu Ala lle Ser Pro Pro Asp Ala Ala Ser Ala Ala
115 120 125
Pro Leu Arg Thr lle Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val
130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala
145 150 155 160
Cys Arg Thr Gly Asp Arg Ala Ala Ala Met Ala Pro Met Thr Gln Thr
165 170 175
Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met lle Asp Glu
180 185 190
. ...
. . ~ ' ~,' . , ' '.' .. ' '" ',.',' ', ' '. ' i, -
. . ...
.: , .
-. - . . .
w o 92/06116 2~9746 il PCT/~S9l/07053
- 39 -
lle lle Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn
195 200 205
Asn Leu Asn Gly Glu Asp Gln Asp lle Leu Met Glu Asn Asn Leu Arg
210 215 220
Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn
225 230 235 240
Ala Ser Ala lle Glu Ser lle Leu Lys Asn Leu Leu Pro Cys Leu Pro
245 250 255
Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile His lle Lys Asp Gly
260 265 270
Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu
275 280 285
Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala lle Phe
290 295 300
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 322 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iil) HYPOTHETICAL: NO
(iv) ANTI SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
A)a Pro Pro Arg Leu lle Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu
1 S 10 15
Leu Glu Ala Lys Glu Ala Glu Asn lle Thr Thr Gly Cys Ala Glu His
Cys Ser Leu Asn Glu Asn lle Thr Val Pro Asp Thr Lys Val Asn Phe
Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Al? Val Glu Val Trp
Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
.
WO 92/06116 PCl/lJS91/07053
-- 40 --
Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
100 105 110
Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala
115 120 125
Pro Leu Arg Thr lle Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val
130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala
145 lSO 155 160
Cys Arg Thr Gly Asp Arg Ala Ala Ala Ser Gly Gly Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala Met Ala Pro Met
180 185 190
Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met
195 200 205
lle Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu
210 215 220
Asp Phe Asn Asn Leu Asn 61y Glu Asp Gln Asp lle Leu Met Glu Asn
225 230 235 240
Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser
245 250 255
Leu Gln Asn Ala Ser Ala lle 61u Ser lle Leu Lys Asn Leu Leu Pro
260 265 270
Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro lle His lle
2~5 280 285
Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu
290 295 300
Lys.Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala
305 310 315 320
Ile Phe
~2) INF0RMATION FOR SEQ ID NO:10:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
WO 92/06116 6 . - - PCr/US91/07053
Zc~;9~4 -i
-- 41 --
(D) TOPOLOGY: l inear
(ii) MOLECULE TYPE: protein
( i ~ i ) HYPOTHETICAL: NO
( tv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ala Pro Met Thr Gln Tnr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
Ser Asn Met lle Asp 61u lle lle Thr His Leu Lys Gln Pro Pro Leu
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp lle Leu
Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala
So 55 60
Val Lys Ser Leu Gln Asn Ala Ser Ala lle Glu Ser lle Leu Lys Asn
Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro
lle His lle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr
100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala lle Phe Leu Glu Ala Ala Ala Ser Leu Pro Ala Met Thr
130 135 140
Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys
145 150 . 155 160
Leu Glu Gln Val Arg Lys lle Gln Gly Asp Gly Ala Ala Leu Gln Glu
165 170 175
Lys Leu Cys Ala Thr Iyr Lys Leu Cys His Pro Glu Glu Leu Val Leu
180 185 190
Leu Gly His Ser Leu Gly 11e Pro Trp Ala Pro Leu Ser Ser Cys Pro
195 ~OO 205
Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
210 215 220
.. .. ... . .: .,. , - . -
~9746 ~ PCT/US91/07053
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly lle Ser Pro
225 230 235 240
Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp~Phe
245 250 255
Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Al~
260 265 270
Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln
275 280 285
Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu
290 295 300
Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
305 310 315
(2) INFORMATION FOR SEQ ID NO:II:
(i) SE~UENCE CHARACTERISTICS:
(A) LENGTH: 994 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A NAME/KEY: CDS
(B LOCATION: 14..977
~x) FEATURE:
~A) NAME~KEY: mat_pept1de
(B) LOCATION: 71..977
(xl) SEQUENCE DESCRIPIION: SEQ ID NO:ll:
AATTGCCGCC ACC ATG AGC CGC CTG CCC GTC CTG CTC CTG CTC CAA CTC q9
Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu
- 19 - 15 - 10
CTG GTC CGC CCC GGA CTC C M GCT CCC ATG ACC CAG ACA ACT AGT TTG 97
Leu V~l Arg Pro Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Ser Leu
1 5
wo 92/06116 z ~ S 9 ~ 4 6 ;` P.cr~69!/070s3
MG ACA AGC TGG GTT MC TGC TCT AAC ATG ATC GAT GM AT~ ATA ACA 145
Lys Thr Ser Trp Val Asn Cys Ser Asn Met lle Asp Glu lle lle Thr
10 1~ 20 25
CAC TTA AAC GAG CCA CCT TTG CCT TTG CTG GAC TTC AAC MC CTC MT 193
His leu Asn Glu Pro Pro leu Pro Leu Leu Asp Phe Asn Asn Leu Asn
30 35 40
GGG GAA GAC CM GAC ATT CTG ATG GM AAT AAC CTT CGA AGG CCA MC 241
Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn
~5 50 55
CTG GAG GCA TTC MC AGG GCT GTC MG AGT TTA CAG AAT GCA TCA GCA 289
Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala
60 65 70
ATT GAG AGC ATT CTT AAA MT C.TC C~G CCA TGT CTG CCC CTG GCC ACG 337
lle Glu Ser lle Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr
75 80 85
GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT 385
Ala Ala Pro Thr Arg His Pro lle His Ile Lys Asp Gly Asp Trp Asn
90 95 100 105
GM TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT GAG MT GCG 433
Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala
110 115 120
CAG GCT CM CAG ACG ACT TTG TCG CTA GCG ATC TTT CTA GAC ATG GCC 48}
Gln Ala Gln G~n Thr Thr Leu Ser Leu Ala lle Phe Leu Asp Met Ala
125 130 135
CCA CCA CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG 529
Pro Pro Arg Leu lle Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu
140 145 150
GAG GCC MG GAG GCC GAG MT ATC ACG ACG GGC TGT GCT GM CAC TGC 577
Glu Ala Lys Glu Ala Glu Asn lle Thr Thr Gly Cys Ala Glu ~is Cys
155 160 165
AGC TTG MT GAG MT ATC ACT GTC CCA GAC ACC MA GTT MT TTC TAC 625
Ser Leu Asn Glu Asn lle Thr Val Pro Asp Thr Lys Val Asn Phe Tyr
170 175 180 185
GCG TGG MG AGG ATG GAG GTC G6C CAG CAG GCC GTA GM 6TC TGG CAG 673
Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln
190 195 200
GGC CTG GCC CTG CTG TCG GM GCT GTC CTG CGG GGC CAG GCC CTG TTG 721
Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu
205 210 215 . ..
~ ~ ` ` ` ` . . ' ` . ~
wo 9~74~ PCl/US91/07053
.. ~
-- 44 --
GTC M C TCG AGC CAG CCG TGG GAG CCC CTG C M CTG CAT GTG GAT AAA 769
Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys
220 225 230
GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT CGG GCT CTG GGA 817
Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly
235 240 245
GCT CAG AAG G M GCC ATC TCC CCT CCA GAT GCG GCC TCA GCT GCT CCA 865
Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro
250 255 260 265
CTC CGA ACA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC CGA GTC TAC 913
Leu Arg Thr lle Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr
270 275 280
TCC M T TTC CTC CGG GGA AAG CTG M G CTG TAC ACA GGG GAG GCA TGC 961
Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys
285 290 295
AGG ACA GGG GAC AGA T GATAAGGATC CG M TTC 994
Arg Thr Gly Asp Arg
300
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1015 base pairs
(B) TYPE: nucleic acid
(C) STRANOEDNESS: double
(D) TOPOL06Y: both
(ii) MOLECULE TYPE: DNA (genomic)
(11t) HYPOTHETICAL: NO
~lv) ANTI SENSE: NO
(1x) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 8..998
(1x) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 89..998
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TCGAGCC ATG GGG GTG CAC GAA TGT CCT GCC TGG CTG TGG CTT CTC CTG 49
Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu
-27 -25 -20 -15
~ .. ., ~ . . : . ;. - :,., ; - ., . . . : - .- ~
. , . - ... - , . : . . . .- .. , .: - . - .. . . .
WO 92/061 16 Z~9746 PCr/US91/07053
.i
-- 45 --
TCC CTG CTG TCG CTC CCT CTG GGC CTC CCA GTC CTG GGC GCC CCA CCA 97
Ser Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro
-10 -5
CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG GAG GCC 145
Arg Leu lle Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala
5 10 15
AAG GAG GCC GAG M T ATC ACG ACG GGC TGT GCT GAA CAC TGC AGC TTG 193
Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu
20 25 30 35
M T GAG M T ATC ACT GTC CCA GAC ACC AAA GTT M T TTC TAC GCG TGG 241
Asn Glu Asn Ile Thr Val Pr~ Asp Thr Lys Val Asn Phe Tyr Ala Trp .
40 45 50
AAG AGG ATG GAG GTC GGC C~.G CAG GCC GTA G M GTC TGG CAG GGC C~G 289
Lys Arg Met Glu Val Gly G~n Gln Ala Val Glu Val Trp Gln Gly Leu
55 60 65
GCC CTG CTG TCG G M GCT GTC CTG CGG GGC CAG GCC CTG TTG GTC M C 337
Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn
70 75 80
TCG AGC CAG CCG TGG GAG CCC CT~ C M CTG CAT GTG GAT AAA GCC GTC 385
Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val
85 90 95
AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT CGG GCT CTG GGA GCT CAG 433
Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln
100 IOS 110 115
M G G M GCC ATC TCC CCT CCA GAT GCG GCC TCA GCT GCT CCA CTC CGA 481
Lys Glu Ala lle Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg
120 125 130
ACA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC CGA GTC TAC TCC AAT 529
Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn
135 140 145 .
TTC CTC CGG GGA AAG CTG AAG CTG TAC ACA GGG GAG GCA TGC AGG ACA 577
Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr .
150 155 160
GGG GAC AGA GCG GCC GCC ATG GCT CCC ATG ACC CAG ACA ACT AGT TTG 625
Gly Asp Arg Ala Ala Ala Met Ala Pro Met Thr Gln Thr Thr Ser Leu
165 170 175 :
AAG ACA AGC TGG GTT MC TGC TCT M C ATG ATC GAT G M ATT ATA ACA 673
Lys Thr Ser Trp Val Asn Cys Ser Asn Met lle Asp Glu lle lle Thr
laO 185 190 195
,. ~ j, . . , !
WO 92/06116 PCr/US91/07053
2(~9746
-- 46 --
CAC TTA MC GAG CCA CCT TTG CCT TTG CTG GAC TTC MC MC CTC MT 721
His Leu Asn Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn
200 205 210
GGG GM GAC CAA GAC ATT CTG ATG GM MT MC CTT CGA AGG CCA MC 769
Gly Glu Asp Gln Asp lle Leu Met Glu Asn Asn Leu Arg Arg Pro Asn
215 220 225
CTG GAG GCA TTC Mt AGG GCT GTC MG AGT TTA CAG MT GCA TCA GCA 817
Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala
230 235 240
ATT GAG AGC ATT CTT MA MT CTC CTG CCA TGT CTG CCC CTG GCC ACG 865
lle Glu Ser lle Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr
245 250 255
GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC MG GAC GGT GAC TGG MT 913
Ala Ala Pro Thr Arg His Pro lle His lle Lys Asp Gly Asp Trp Asn
260 265 270 275
GM TTC CGG AGG A M CTG ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG 961
Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala
280 285 290
CAG GCT CM CAG ACG ACT TTG TCG CTA GCG ATC TT~ T AGTAAGGATC 1008
Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala lle Phe
295 300
CGMTTC 1015
(2) INFORMATION FOR SEQ ID NO:13:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1039 base pairs
(B) TYPE: nucle~c ac~d
(l STRAMDED~ESS: doub7e
~D TOPOLO&Y: both
(11) MOLECULE TYPE: DNA (genom~c)
(lil) HYPOTHETICAL: NO
(lv) ANTI SEHSE: NO
(~x) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 14..1021
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 71..1021
~ . .~ - .......... . . . . . . .
' :- '. . " `'" , ' ;' ' . ' , ' ', '; . ' . . ';' . , , , ~ ~ .
W o 92/06116 2 ~i9 7 4 6 PCT/US91/07053
- 47 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
AATTGCCGCC ACC ATG AGC CGC CTG CCC GTC CTG CTC CTG CTC CM CTC 49
Met Ser Arg Leu Pro Val leu Leu Leu Leu Gln Leu
- I 9 - 1 5 - 1 o
CTG GTC CGC CCC GGA CTC CM GCT CCC ATG ACC CAG ACA ACT AGT TTG 97
Leu Val Arg Pro Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Ser Leu
-5 1 5
AAG ACA AGC TGG GTT MC TGC TCT MC ATG ATC GAT GM ATT ATA ACA 145
Lys Thr Ser Trp Val Asn Cys Ser Asn Met lle Asp Glu lle lle Thr
10 15 20 25
CAC TTA MC GAG CCA CCT TTG CCT TTG CTG GAC TTC MC MC CTC MT 193
His Leu Asn Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn
30 35 40
GGG GM GAC CAA GAC ATT CTG ATG GM AAT MC CTT CGA AGG CCA AAC 241
Gly Glu Asp Gln Asp lle Leu Met Glu Asn Asn Leu Arg Arg Pro Asn
45 So SS
CTG 6AG GCA TTC MC AGG GCT GTC MG AGT TTA CAG MT GCA TCA GCA 289 . . .
Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala
60 65 70
ATT GAG AGC ATT CTT AAA MT CTC CTG CCA TGT CTG CCC CTG GCC ACG 337
lle Glu Ser lle Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr
75 80 85 :
GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT 385
Ala Ala Pro Thr Arg His Pro lle His lle Lys Asp Gly Asp Trp Asn
GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG MA ACC CTT GAG AAT GCG 433
Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala
110 llS 120
CAG GCT CM CAG ACG ACT TTG TCG CTA GCG ATC TTT CTA GAA GCG GCC 481
Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala lle Phe Leu Glu Ala Ala
125 130 ~35
GCA AGC TTA CCT GCC ATG ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC 529
Ala Ser Leu Pro Ala Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro
140 145 150
CAG AGC TTC CTG CTC AAG TGC TTA GAG CAA GTG AGG MG ATC CAG GGC 577
i~ Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys lle Gln Gly
155 160 165
GAT GGC GCA GCG CTC CAG GAG AAG CTG TGT GCC ACC TAC AAG CTG TGC 625
Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
170 175 180 185
WO 92/061 16 - PCI`/US91/07053
ZC~G971~6 - 48 -
CAC CCC GAG GAG CTG GTG CTG CTC GGA CAC TCT CTG GG~ ATC CCC TGG 673
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp
190 l9S 200
GCT CCC CTG AGC TCC ~GC CCC AGC CAG GCC CTG CAG CTG GCA GGC TGC 721
Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys
205 210 215
TTG AGC CM CTC CAT AGC GGC CTT TTC CTC TAC CAG GGG CTC CTG CAG 769
Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln
220 225 230
GCC CTG GM GGG ATA TCC CCC GAG TTG GGT CCC ACC TTG CAC ACA CTG 817
Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu His Thr Leu
235 240 245
CAG CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GM 865
Gln Leu Asp Val Ala Asp Phe Ala Thr Thr lle Trp Gln Gln Met Glu
250 255 260 265
GAA CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG 913
Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro
270 275 280
GCC TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG GTC CTG GTT GCT 961
Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala
285 290 295
AGC CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC CGC GTT CTA CGC CAC 1009
Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
300 305 310
CTT GCG CAG CCC TGATMGGAT CCGMTTC 1039
Leu Ala Gln Pro
315
(2) INFORMATION FOR SEQ ID NO:14:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: IOSI base pairs
B) TYPE: nucleic acid
C STRANDEDNESS: doub le
D TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic)
( ~ i i ) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(~x) FEATURE:
- , . ; . ;,; , , . . . 1 " :, -. - . . :i . .. . .
WO 92/06116 PCI/US91/07053
~ z(~69'74~i .
- 49 -
(A) NAME/KEY: CDS
(B) LOCA~ION: 14..1033
(ix) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 71..1033
(xi) SE~UENCE DESCRIPTiON: SEQ ID NO:14:
M TTGCCGCC ACC ATG AGC CGC CTG CCC GTC CTG CTC CTG C~C C M CTC 49
Met Ser Ar~ Leu Pro Val Leu Leu Leu Leu Gln Leu -
_19 -Is -10
CTG GTC CGC CCC GGA CTC CI~A GCT CCC ATG ACC CAG ACA ACT AGT TTG 97 . .
Leu Val Arg Pro Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Ser Leu
-5 1 5
~AG ACA AGC TGG GTT M C TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA 145
Lys Thr Ser Trp Val Asn Cys 5~r Asn Met lle Asp Glu lle lle Thr
10 15 20 25
CAC TTA AAC GAG CCA CCT TTG CCT TTG CTG GAC TTC AAC M C CTC MT 193 : :
His Leu Asn Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn
30 35 40
GGG G M GAC C M GAC ATT CTG ATG G M AAT MC CTT CGA AGG CCA MC 241
Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn
45 50 55
CTG GAG GCA TTC M C AGG GCT GTC M G AGT TTA CAG M T GCA TCA GCA 289
Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala
60 65 70
ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG 337
Ile Glu Ser lle Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr
75 80 85
GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG M T 385
Ala Al~ Pro Thr Arg Hts Pro lle His Ile Lys Asp Gly Asp Trp Asn
90 95 100 105
G M TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT GAG M T GCG 433
61u Phe Arg Arg Lys leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala
110 115 120
CAG GCT CAA CA6 ACG ACT TTG TCG CTA GCG TCC GGA GGC GGT GGC TCG 481
Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala Ser Gly Gly Gly Gly Ser
125 130 135
GGC GGT GGC GGC TCG GGT GGC GGC GGC TCT GCG CTA GCG ATC TTT CTA 529
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Ala lle Phe Leu
140 145 150
., . . `. :, :. : . . , . .. ,, : ..
~VO 92/06116 ,, ~ ~, PCr/US91/07053
-- 50 --
GAC ATG GCC CCA CCA CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG 577
Asp Met Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg
155 160 165
TAC CTC TTG GAG GCC MG GAG GCC GAG MT ATC ACG ACG GGC TGT GCT 625
Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn lle Thr Thr Gly Cys Ala
170 175 180 185
GM CAC TGC AGC TTG MT GAG MT ATC ACT GTC CCA GAC ACC MA GTT 673
Glu His Cys Ser Leu Asn Glu Asn lle Thr Val Pro Asp Thr Lys Val
190 195 200
AAT TTC TAC GCG TGG MG AGG ATG GAG GTC GGC CAG CAG GCC GTA GM 721
Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu
205 210 215
GTC TGG CAG GGC CTG GCC CTG CTG TCG GM GCT GTC CTG CGG GGC CAG 769
Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln
220 225 230
GCC CTG TTG GTC AA~ TCG AGC CAG CCG TGG GAG CCC CTG CM CTG CAT 817
Ala Leu Leu \~al Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His
235 240 245
GTG GAT AM GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT CGG 865
Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg
250 255 260 265
6CT CTG GGA GCT CAG MG GM GCC ATC TCC CCT CCA GAT GCG GCC TCA 913
Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser
270 275 280
GCT GCT CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC CGC MM CTC TTC 961
Ala Ala Pro Leu Arg Thr lle Thr Ala Asp Thr Phe Arg Lys Leu Phe
285 290 295
CGA GTC TAC TCC AAT TTC CTC CGG GGA MG CTG MG CTG TAC ACA GGG 1009
Arg V~l Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly
300 305 310
GAG GCA TGC AGG ACA GGG GAC AGA TGATMGGAT CCGMTTC 1051
Glu A3~5 Cys Arg Thr Gly 3A2p Arg
(2) INFORMATION FOR SEq ID NO:15:
(i) SEqUENCE CHARACTERISTICS:
(A) LENGTH: 1072 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic)
- : ; . . , .
. - . - . . .. - ... ~ . - . - . . -
WO 92/06116 PCl/US91/07053
Z~g~4
-- 51 --
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 8..1054
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 89..1054
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
TCGAGCC ATG GGG GTG CAC G M TGT CCT GCC TGG CTG TGG CTT CTC CTG 49
Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu
-27 -25 -20 -15
TCC CTG CTG TCG CTC CCT CTG GGC CTC CCA GTC CTG GGC GCC CCA CCA 97
Ser Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro
-10 -5
CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG GAG GCC 145
Arg Leu lle Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala
5 10 15
M G GAG GCC GAG M T ATC ACG ACG GGC TGT GCT G M CAC TGC AGC TTG 193
Lys 61u Ala Glu Asn lle Thr Thr Gly Cys Ala Glu His Cys Ser Leu
20 25 30 35
M T GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT M T TTC TAC GCG ~GG 241
Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp
40 45 50
M G AGG ATG GAG GTC GGC CAG CAG GCC GTA G M GTC TGG CAG GGC CTG 289
Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu
GCC CTG CTG TCG G M GCT GTC CTG CGG GGC CAG GCC CTG TTG GTC AAC 337
Ala Leu Leu Ser Glu .~la Val Leu Arg Gly Gln Ala Leu Leu Val Asn
70 75 80
TCG AGC CAG CCG TGG GAG CCC CTG C M CTG CAT GTG GAT MM GCC GTC 385
Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu H~s Val Asp Lys Ala Val
85 90 95
AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT CGG GCT CTG GGA GCT CAG 433
Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln
100 105 110 115
.. , . . . . , . - . . . . -
... .... - .. , . . . . ... - . . . .
- . .
. . . . .~ .
WO 92/06116 PCl/US91/07053
2~69746 - 52 -
MG GM GCC ATC TCC CCT CCA GAT GCG GCC TCA GCT GCT CCA CTC CGA 481
Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg
~20 125 130
ACA ATC ACT GCT GAC ACT TTC CGC MA CTC TTC CGA GTC TAC TCC MT 529
Thr lle Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn
135 140 145
TTC CTC CGG GGA MG CTG MG CTG TAC ACA GGG GAG GCA TGC AGG ACA 577
Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr
.150 155 160
GGG GAC AGA GCG GCC GCC TCC GGA GGC GGT GGC TCG GGC GGT GGC GGC 625
Gly Asp Arg Ala Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
165 170 175
TCG GGT GGC GGC GGC TCT GCG GCC GCC ATG GCT CCC ATG ACC CAG ACA 673
Ser Gly Gly Gly Gly Ser Ala Ala Ala Met Ala Pro Met Thr Gln Thr
180 185 190 195
ACT A6T TTG AAG ACA AGC TGG GTT MC TGC TCT AAC ATG ATC GAT GM 721
Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu
200 205 210
ATT ATA ACA CAC TTA MC GAG CCA CCT TTG CCT TTG CTG GAC TTC MC 769
lle lle Thr His Leu Asn Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn
215 220 225
AAC CTC MT GGG GAA GAC CM GAC ATT CTG ATG GAA MT MC CTT CGA 817
Asn Leu Asn Gly Glu Asp Gln Asp lle Leu Met Glu Asn Asn Leu Arg
230 235 240
AGG CCA MC CTG GAG GCA TTC MC AGG GCT GTC MG AGT TTA CAG MT 865
Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn
245 250 255
GCA TCA 8CA ATT GAG AGC ATT CTT AAA MT CTC CTG CCA TGT CTG CCC 913
Al~ S~r Al~ lle Glu Ser lle Leu Lys Asn Leu Leu Pro Cys Leu Pro
260 265 270 275
CTG GCC ACG GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC MG GAC GGT 961
Leu Ala Thr Ala Ala Pro Thr Arg His Pro lle His lle Lys Asp Gly
280 285 290
GAC TGG MT GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG MM ACC CTT 1009
Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu
295 300 305
GAG MT GCG CAG GCT CM CAG ACG ACT TTG TCG CTA GCG ATC TTT 1054
Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala lle Phe
310 315 320
TAGTMGGAT CCGAATTC 1072
. '.':' `' ''.''~'`'' .:. `,''-'''''''''',.'.~'';,'','" ~.'"' ';'-'.' '''''
W O 92/06116 Z~9746 PC~r/~S91/07053
~,
-- 53 -- :
(2) INFORMATION FOR sEq ID NO:16:
( i ) SEQUENCE CHARACTERISTICS:
( A ) L EHGTH: 429 ba se pa i rs
(B) TYPE: nucleic acid
(C) STRANCEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic)
( i i i ) HYPOTHETICAL: NO
( iv) ANTI-SENSE: NO
( ix) FEATURE:
(A) NAME/KEY: C~S
(B) LOCATION: 10..411
( ix) fEATURE:
(A) NAME/KEY: mat_pept ide
(B) LOCATION: 13..411
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
AAGCTTACC ATG GCT CCC ATG ACC CAG ACA ACT AGT TTG MG ACA AGC 48
Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser
-1 1 5 10
TGG GTT MC TGC TCT MC ATG ATC GAT GM ATT ATA ACA CAC TTA MC 96
Trp Val Asn Cys Ser Asn Met lle Asp Glu Ile lle Thr His Leu Asn
15 20 25
GAG CCA CCT TTG CCT TTG CTG GAC TTC MC MC CTC MT GGG GM GAC 144
Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp
30 35 40
CM GAC ATT CTG ATG GAA MT MC CTT CGA AGG CCA AAC CTG GAG GCA 192
Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala
45 50 5s 60
TTC AAC AGG GCT GTC MG AGT TTA CAG AAT GCA TCA GCA ATT GAG AGC 240
Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala lle Glu Ser
65 70 75
ATT CTT MM AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG GCC GCA CCC 288
lle Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro
80 85 90
ACG CGA CAT CCA ATC CAT ATC MG GAC GGT GAC TGG AAT GM TTC CGG 336
Thr Arg His Pro lle H~s lle Lys Asp Gly Asp Trp Asn Glu Phe Arg
9S 100 105
.... . . . - . . , - . .. . . .
' ' ~ ' " ' :. ", -,; -' , -
.~ ,. . .. . . .
. . ~ . .
- .- .: ~ . ,~. . -,:
wo 92/06116 Pcrt~s9l/07053
2~;97~6
AGG MA CTG ACG TTC TAT CTG MA ACC CTT GAG MT GCG CAG GCT CM 384
Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln
110 115 120
CAG ACG ACT TTG TCG CTA GCG ATC TTT TAGTMGGAT CCGMTTC429
Gln Thr Thr Leu Ser Leu Ala lle Phe
125 130
(2) INFORMATION FOR SEQ ID NO: 17:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 532 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNE SS: doub l e
(D) TOPOLOGY: both
(ii3 MOLECULE TYPE: DNA (genomic)
( i i i ) HYPOTHETICAL: NO
( i v ) ANT I - SENSE: NO
( ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 14..514
( ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 17..514
(x~) SEQU~NCE DESCRIPTION: SEQ 10 NO:17:
AAGCTTACCT GCC ATG GCC CCA CCA CGC CTC ATC TGT GAC AGC CGA GTC 49
Met Ala Pro Pro Arg Leu lle Cys Asp Ser Arg Val
CTG GAG AGG TAC CTC TTG GAG GCC MG GAG GCC GAG MT ATC ACG ACG 97
Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn lle Thr Thr
IS 20 25
GGC TGT GCT GAA CAC TGC AGC TTG MT GAG AAT ATC ACT GTC CCA GAC 145
Gly Cys Ala Glu H1s Cys Ser Leu Asn Glu Asn lle Thr Val Pro Asp
30 35 40
ACC MA GTT AAT TTC TAC GC6 TGG MG AGG ATG GAG GTC GGC CAG CAG 193
Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln
45 50 55 .:
GCC GTA GM GTC TG6 CAG GGC CTG GCC CTG CTG TC6 GAA GCT GTC CTG 241
Ala Yal Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu
60 65 70 75
~ " ; . , . . ........ ... ,.. , . ~. -.. , ~ . . .
~: ' ' ' ' ! . ,
WO 92/061 16 PCr/~;S91/070~3
2C~6~746 : ~-
- 55 -
CGG GGC CAG GCC CTG TTG GTC M C TCG AGC CAG CCG TGG GAG CCC CTG 28
Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu
80 85 90
CAA CTG CAT GTG GAT A M GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT 337
Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr
95 100 105
CTG CTT CGG GCT CTG GGA GCT CAG A~G G M GCC ATC TCC CCT CCA GAT 385
Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala lle Ser Pro Pro Asp
110 llS 120
GCG GCC TCA GCT GCT CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC CGC 433
Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg
125 130 135
AAA CTC TTC CGA GTC TAC TCC M T TTC CTC CGG GGA M G CTG M G CTG 481
Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu
140 145 150 155
TAC ACA GGG GAG GCA TGC AGG ACA GGG GAC AG ATGAT M GGA TCCG M TTC ~32
Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp
160 165
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 556 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ll) MOLECULE TYPE: DNA (genomlc)
(llt) HYPOTHETICAL: NO
(lvJ ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 14..538
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 17..538
(xi) SE~UENCE DESCRIPTION: SEQ ID NO:18:
M GCTTACCT GCC ATG ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC CAG ~9
Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
-1 1 5 10
- . . . .
, : : . - . . , , . .. : " . . . :
: .. , . . - . . . ... . . . .. ...
. . .. - .,.:.. ~;: .. . .
WO 92/06116 . PCr/US91/07053
2(:~9'~6
-- 56 --
AGC TTC CTG CTC MG TGC TTA GAG CM GTG AGG MG ATC CAG GGC GAT 97
Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys lle Gln Gly Asp
15 20 25
GGC GCA GCG CTC CAG GAG MG CTG TGT GCC ACC TAC MG CTG TGC CAC 145
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His
30 35 40
CCC GAG GAG CTG GTG CTG CTC GGA CAC TCT CTG GGC ATC CCC TGG GCT 193
Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly lle Pro Trp Ala
45 50 55
CCC CTG AGC TCC TGC CCC AGC CAG GCC CTG CAG CTG GCA GGC TGC TTG 241
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
60 65 70 75
AGC CM CTC CAT AGC GGC CTT TTC CTC TAC CAG GGG CTC CTG CAG GCC 289
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
80 85 90
CTG GM GGG ATA TCC CCC GAG TTG GGT CCC ACC TTG CAC ACA CTG CAG 337
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu His Thr Leu Gln
95 100 105
CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA GM 385
Leu Asp Val Ala Asp Phe Ala Thr Thr lle Trp Gln Gln Met Glu Glu
110 115 120
CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG GCC 433
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
125 130 135
TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG GTC CTG GTT GCT AGC 481
Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
140 145 150 155
CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC CGC GTT CTA CGC CAC CTT 529
Hls Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
160 165 170
GCG CAG CCC TGATAAGGAT CCGAATTC 556
Ala Gln Pro
(2) INFORMATION FOR SEQ ID NO:l9:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEONESS: single
(D) TOPOLOGY: l inear
(1i) MOLECULE TYPE: DNA
.. . . ; , : - . . . ~: . . -
., , , ... ' - .
WO 92/061 16 2i[~9746 PCI /US91/07053
,.. ` .
- 57 -
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
M TTGCCGCC ACCATGAGCC GCCTGCCCGT CCTGCTCCT 39
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: SO hase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GCTCCAACTC CTGGTCCGCC CCGGACTCCA AGCTCCCATG ACCCAGACAA SO
(2) INFORMATION FOR SEQ ID NO:21.
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B TYPE: nuclelc acid
(C STRANDEDNESS: slngle
(D TOPOLOGY: llnear
(li) MOLECULE TYPE: DNA
(lll) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
(x1) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CTAGTTGTCT GGGTCATGGG AGCTTGGAGT CCGGGGCGG 39
` . ' . "~ , , ., , '
. . ' , . ., : "
' " ' . ~ , ' ' ' ', ' , '' . ':
WO 92/061 16 - . - PCr/US91/07053
. ,
Z~ 9746 - 58 -
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SE~UENCE DESCRIPTION: SEQ ID NO:22:
ACCAGGAGTT GGAGCAGGAG CAGGACGGGC AGGCGGCTCA TGGTGGCGGC 50
(2) INFORMATION FOR SEQ 10 NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs ;
(8) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(111) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CTA6CGATCT TTCTAGA 17
(2) INFORMATION FOR SEQ ID NO:2q:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucle~c acid
(C) STRAHDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: DNA
(lii) HYPOTHETICAL: NO
... .. . .. . ........ . . . . .
. . , . .. . . ..
- . ~ . . . - . .. . . .
w o 92/06116 za~97~6 PCT/~S91/07053
.~, ., . . ~ i
- 59 -
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CATGTCTAGA M GATCG 17
(2) INfORMATlON FOR SEq ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CTAGCGTCCG GAGGCGGTGG CTCGGGCGGT GGCGGCTCGG GTGGCGGCGG CTCTGCG 57
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS-
(A) LENGTH: 57 base palrs
(B) TYPE: nuclelc acid
C) STRANDEDNESS~ slngle
O) TOPOLOGY: llnear
(l1) MOLECULE TYPE: DNA
~lli) HYPOTHETICAL: NO
(l~) ANTI-SENSE: NO
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CTAGCGCAGA GCCGCCGCCA CCGCAGCCGC CACCGCCCGA GCCACCGCCT CCGGACG 57
(2) INFORMATlON FOR SEQ ID NO:27:
(1) SEQUENCE CHARACTERISTICS
(A) LENGTH: 15 amlno acids
. .. . .. . . . , 1
. . .
, .. , . ~ ~ ~ . .
.. , ., .... . ... .. ~ - : .. . .
, . ... . . ~ .
..... , ,~. ~,,, . . ., , ... -. - .. . . . . .
WO 92/06116 ~ ` PCI/llS91/07053
2~;974~
- 60 -
(B) TYPE: amino acid
(C) STRANOEDNESS: single
(D) TOPOlOGY: linear
(li) MOLECULE TYPE: peptide
(iil) HYPOTHETICAL: HO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nuclelc acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(lii) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:28:
TTGTCGCTAG CGTCCGGAGG C 21
(2) INFORMATION FOR SEQ ID NO:29:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base palrs
(B) TYPE: nuclelc acld
(C) STRANOEDNESS: slngle
(D) TOPOLOGY: linear . .
(ll) MOLECULE TYPE: DNA
(lll) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
.
, - - . . . . . . . . .
'. ` ,, :, : '. ' '' ' ., ' ' .. ' '' '..... - ' :
wo 92/06116 PCI/US91iO7053
~ 2~97~6
- 61 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CTAGM GCGG CCGCA 15
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleie acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
TTCGCCGGCG TTCGA 15
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nuclelc acid
~C STRANDEDNESS: single
(D TOPOLOGY: llnear
(ll) MOLECULE TYPE: DNA
(lli) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:31:
TCGAGCCATG GGGGTGCACG AATGTCCT 28
(2) INFORMATION FOR SE~ ID NO:32:
(l) SE~UENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
WO 92/06116 . . PCI`/US91/070
z c~jg~7~LÇ; - 62 -
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
GCC~GGCTGT GGC~CTCCT GTCCCTGCTG TC 32
(2) INFOR~A~ION EOR SE~ IO ~0:33: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(O) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(x1) SEQUENCE DESCRIPTION: SEQ ID NO:33:
GCTCCCTCTG GGCCTCCCAG TCCTGGGCTG CA 32
(2) INFORMATION FOR SEQ ID NO:34:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pa1rs
B) TYPE: nucleic acid
C) STRANOEDNESS: s1ngle
O) TOPOLOGY: l~near
(ii) MOLECULE TYPE: DNA
(1i1) HYPOTHETICAL: NO
(~v) ANTI-SENSE: NO
(xi) SEQUENCE OESCRIPTION: SEQ ID NO:34:
6CCCAGGACT GGGAGGCCCA GAGGGA 26
W o 92/06116 Z~i97~L6 PCT/US91/07053
., -. ,,, - ,
- 63 -
(2) INFORMATION FOR SEQ ID NO:35:
(i) sEquENcE CHARACTERISTICS:(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
GCGACAGCAG GGACAGGAGA AGCCACAGCC AGGCAGGACA TT 42
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(1~i) HYPOTHETICAL: NO
(~v) ANTI-SENSE: NO
(xl) SEQUENCE DESCRlPTION: SEQ ID NO:36:
CGTGCACCCC CATGGC 16
(2) INFORMATION FOR SEQ ID NO:37:
(t) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANOEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA
(lll) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
WO 92/06116 - . ?~ PCr/US91/070~3
~,
2C~j9~744i - 64 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
GCCCCACCAC GCCTCATCTG T 21
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENG~H: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear :
(ii) MOLECULE TYPE: DNA -
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
G M TTCGGAT CCTTATCATC T 21
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 30 base pairs
B) TYPE: nucleic acld
(C) STRANDEDNESS: single
(D) TOPOLOGY: ltnear
(11) MOlECULE TYPE: ONA
~1l1) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
(x1) SEQUENCE DESCRIPTION: SEQ ID NO:39:
CTAGTCTCTA GAATGGGGGT CCACGAATGT io : .
~2) INFORMATION FOR SEQ ID NO:40: ~.
(1) SE~UENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
B) TYPE: nucleic acid
C) STRANDEDNESS: single . : .
(D) TOPOLOGY: linear
.
WO 92/061~6 2C~;974~PCI/US91/070~3
65 -
(ii) MOLECULE TYPE: DNA
(iii) HYPO~HETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
AGCCATGGCG GCCGCTCTG~ CCCCTGTCCT 30
(2) INFORMATION FOR SEQ Il) NO:41:
(i) SEQUENCE CHARACTcRISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(x1) SEQUENCE DESCRIPTION: SEQ ID NO:41:
6ACAGAGCGG CCGCCATGGC TCCCATGACC 30
~2) INFORMATION FOR SEQ ID NO:42:
(1) SEqUENCE CHARACTERISTICS:
A) LENGTH: ~0 base pairs
B TYPE: nucle1c acid
C STRANDEDNESS: s1ngle
(D) TOPOLOGY: linear
(1i) MOLECULE TYPE: DNA
(~11) HYPOTHETICAL: NO
(1v) ANTI-SENSE: NO
(xl) SEQUENCE DESCRIPTION: SEQ ID NQ:42:
G M TTCGGAT CCTTACTA M AGATCGCTAG 30
- . - - ~. - : . . . .. . .. ... . . - . -;
,,.. .. ,.. . ~ . . , . , , .- ; .. , . .. - - . . . .. . .
WO 92/06116 . ~ PCI'/US91/070~3
Zc~9~46 - 66 -
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) AHTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
GGCCGCTTCC GGAGGCGGTG GCTCGGGCGG TGGCGGCTCG GGTGGCGGCG GCTCTGC 57
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:44:
GGCCGCAGAG CCGCCGCCAC CCGAGCCGCC ACCGCCCGAG CCACCGCCTC CGGCAGC , 57
