`Nagata et al.
`
`III IIHIIII
`
`USOO5574136A
`Patent Number:
`11
`(45) Date of Patent:
`
`5,574,136
`Nov. 12, 1996
`
`54) DNA ENCODING GRANULOCYTE
`COLONY-STMULATING FACTOR
`RECEPTOR AND PROTEIN THEREOF
`
`75 Inventors: Shigekazu Nagata, Suita; Rikiro
`Fukunaga, Minoo, both of Japan
`(73) Assignee: Osaka Bioscience Institute, Japan
`21) Appl. No.:
`923,976
`22) PCT Filed:
`Mar. 22, 1991
`86 PCT No.:
`PCT/JP91/00375
`S371 Date:
`Sep. 22, 1992
`S 102(e) Date: Sep. 22, 1992
`(87) PCT Pub. No.: WO91/14776
`PCT Pub. Date: Oct. 3, 1991
`Foreign Application Priority Data
`(30)
`Mar. 23, 1990 (JP)
`Japan ...................................... 2-74539
`Jul. 3, 1990 (JP)
`Japan .................................... 2-176629
`(51) Int. Cl." ........................ C12N 15/12; C07K 14/705;
`CO7K 141715
`52 U.S. C. ........................ 530/350; 536/23.1; 536/23.5;
`435/69.1; 530/351
`58 Field of Search ................................ 435/69.1, 240.2,
`435/252.3, 320.1; 530/350, 351; 536/23.5,
`23.52
`
`56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,675,285 6/1987 Clark et al. .......................... 435/1723
`
`5,422,248 6/1995 Smith et al. ........................... 435/69.
`FOREIGN PATENT DOCUMENTS
`WO91/05046 4/1991 European Pat. Off..
`OTHER PUBLICATIONS
`Yamasaki et al, Science, 241, 1988, pp. 825-828.
`Sims et al, Science, 241, 1988, pp. 585-589.
`Uzumaki et al., PNAS, 86, 1989, pp. 9323–9326.
`Cosman et al., 1990, vol 2(1) pp. 1-31.
`Larsen, A. et al., J. Exp. Med., The Rockefeller Univ. Press.
`vol. 172 "Expression cloning of a human granulocyte colo
`ny-stimulating factor receptor: a structural mosaic of
`hematopoietin receptor, immunoglobulin, and fibronectin
`domains", pp. 1559-1570 (Dec. 1990).
`Park, L. S. et al., Blood, vol. 74, No. 1, "Interleukin-3,
`GM-CSF, and G-CSF, receptor expression on cell lines and
`primary leukemia cells: receptor heterogeneity and relation
`ship to growth factor responsiveness' pp. 56-65, (Jul. 1989).
`Primary Examiner-Marianne P. Allen
`(57)
`ABSTRACT
`A murine G-CSF receptor cDNA was cloned from a cDNA
`library prepared from mouse myeloid leukemia cells and the
`structure analyzed. A human G-CSF receptor cDNA was
`then cloned from cDNA libraries constructed from human
`placenta cells or human histocytic lymphoma cells using the
`murine G-CSF receptor cDNA as a probe, the structure
`analyzed and expressed in a host cell transformed with it.
`The stable production of human G-CSF receptor can be
`accomplished by transforming a host cell with the cloned
`G-CSF receptor cDNA and making the transformant express
`the G-CSF receptor.
`
`5 Claims, 21 Drawing Sheets
`
`Bracco Ex. 2009
`Maia v. Bracco
`IPR2019-00345
`
`1
`
`
`
`US. Patent
`
`Nov. 12, 1996
`
`Sheet 1 of 21
`
`5,574,136
`
`U]
`
`0<0
`
`=00
`
`00.<
`
`0:
`
`3.Us
`
`<00
`
`m0<
`
`0<0
`
`.000
`
`0.0<
`
`0000.
`
`0000
`
`20
`
`0<0
`
`:00
`
`0<0
`
`0000
`
`0.00
`
`8.0
`
`<00
`
`02
`
`00.0
`
`00>
`
`:3<00
`
`00.<
`
`0o:
`
`0.00
`
`8.0
`
`08
`
`0a
`
`000
`
`00<
`
`0<0
`
`:00
`
`0.00
`
`2.0
`
`0<<
`
`3‘
`
`00.0
`
`:3
`
`0.<0
`
`m0:
`
`00<
`
`000.
`
`.000.
`
`0.0.0
`
`03
`
`08
`
`0a
`
`0.00.
`
`0.00
`
`0.<<
`
`o:
`
`<<0
`
`20.0
`
`00
`
`cm<
`
`00.<
`
`:00
`
`.3000
`
`:300.0
`
`00.0.
`
`<000.00.
`
`000000
`
`000
`
`<<00<0
`
`.503
`
`.308
`
`000.
`
`000
`
`0<0
`
`30
`
`0.00
`
`00<
`
`<00
`
`8.0
`
`0<0
`
`:00
`
`0....
`
`.3.
`
`.3
`
`0<0
`
`20
`
`000
`
`2.0
`
`<00
`
`0w0<
`
`00m<:30<000.0
`
`0.00.0<0
`
`000:00
`
`00<
`
`000
`
`.3000
`
`000
`
`00.
`
`.5
`
`as
`
`80
`
`.00
`
`00.<
`
`0:
`
`<<0
`
`50
`
`000
`
`000
`
`m2
`
`0<0
`
`50
`
`as.3as.000
`
`0.00.
`
`000as
`
`.3.00
`
`000
`
`.00
`
`00.0
`
`00.00
`
`00.0
`
`000
`
`<5.00<000.0<<<00
`
`00.0wollm0005008.0
`
`<<000<
`0000<00.00
`
`E00000.
`000.032.0
`
`00.000.0
`
`00<03
`
`0<0000.
`
`£0£0
`
`80
`
`8..Us
`
`.000....
`
`.08
`
`0s
`
`<<0
`
`50
`
`000
`
`.3
`
`00.0
`
`000.00
`
`<<000.0
`
`5000>
`
`000
`
`00.000<
`
`:3000
`
`.00080
`
`0.00.0
`
`00.4.
`
`o:
`
`000
`
`00<
`
`.5.3.08.000
`
`00.<
`
`0e:
`
`000
`
`80
`
`000000.
`000000
`.300.00<00.00.
`:003.0
`
`00.0
`
`00
`
`00:
`
`00000<
`
`80.50
`
`00<00.0
`
`00000>
`
`0<0
`
`:00
`
`000.
`
`000.
`
`<0<
`
`0.00.
`
`:000o:
`
`<<000.<
`
`0<0.
`
`E0.80
`
`0000
`
`00<0.00
`
`20000
`
`000<00
`
`.00080
`
`0.0<00000<0.000000<0<00000.0000000000<<0<00<0<0<0<<0<0<0<00<0<0
`
`2.0
`
`.00
`
`00..
`
`3.
`
`00<
`
`.80
`
`00.<
`
`0:
`
`:300.0
`
`05
`
`.0.
`
`00<
`
`.50
`
`0<<
`
`cm<
`
`0<0
`
`:00
`
`0.0
`
`.00
`
`Us
`
`02.
`
`00<
`
`0000.
`
`0.0.0
`
`000
`
`0<0
`
`:00
`
`0<00.0<
`
`0<00<<
`
`00000<
`
`000
`
`00.0
`
`00<
`
`$0
`
`000.
`
`.000.
`
`.305
`
`0<<
`
`00.0
`
`000.
`
`.80
`
`000.
`
`30
`
`.30.00
`
`0.00
`
`20
`
`0<0
`
`30
`
`:300.0.
`
`0000.
`
`0.<0
`
`0000
`
`00m
`
`<<0
`
`:00
`
`0<0
`
`20
`
`so
`
`.538.0-03
`
`0<000.00.0<<0<000
`
`
`00.0<0.0000.00.0.00.<00.000<
`
`000
`
`00<
`
`20.0
`
`000.
`
`000
`
`0<0
`
`E0
`
`000.
`
`.000.
`
`00<
`
`0000.
`
`:300.0
`
`00<
`
`9.0
`
`.00
`
`<<<
`
`00.0
`
`000
`
`000.
`
`.000.
`
`0.00
`
`0:.
`
`000
`
`00<
`
`.3000
`
`00.0
`
`00>
`
`<00
`
`20
`
`00.0
`
`:3
`
`0.00
`
`20
`
`0.0.0
`
`00>
`
`000
`
`0<<
`
`m3
`
`0<0
`
`0000
`
`000
`
`00.0
`
`:3
`
`<<0
`
`0<<
`
`0<<
`
`.0000.300.0
`
`000
`
`8..
`
`0.<0
`
`00<
`
`.000
`
`0.0
`
`30
`
`00.0.
`
`0.00.
`
`000
`
`0<0
`
`£0
`
`000
`
`
`
`.2.,-.30.00
`
`00.0
`
`00>
`
`<00
`
`00
`
`.2.380000
`
`00.0
`
`0.000
`
`0.00
`
`8.0
`
`00<
`
`0<0
`
`0000.0.0
`
`030a
`
`0.00
`
`2.0
`
`<<0
`
`:00
`
`0:
`
`.000.00
`
`0.0.3
`
`.00..000
`
`3..3
`
`00<00<
`
`00000<
`
`000.0<<
`
`:300<
`
`000.
`
`000
`
`000.
`
`900
`
`00.0.
`
`30
`
`<<<
`
`0.00
`
`000.
`
`30
`
`000.
`
`00<
`
`0»:
`
`000
`
`000
`
`00<
`
`.80
`
`00.0
`
`0.000
`
`<00.
`
`.80
`
`0.00.
`
`000
`
`00.0
`
`:3
`
`00.0
`
`:3
`
`000
`
`00<
`
`000.
`
`000.
`
`0000
`
`<<<0000.00<<000.00<<0.00<000<00<000.0<<000.<00.0<<0.0<<00.000<<0<000<0<000<<<0<000<<<000E00000<<00.000.<0.000.0<00.<0<0<00.0000.<0
`
`_00m
`
`0000.
`
`00<
`
`00.<
`
`.3000
`
`00080
`
`.320
`
`000:300000.0
`
`0000<0
`
`2030
`
`0s
`
`02
`
`00.0
`
`0m>
`
`<<0
`
`50
`
`00.
`
`000.000
`0<000<
`
`3000<
`
`
`
`£0Eflmmg.3
`
`.3
`
`000.<000<<<00.
`
`00000<
`8008
`
`000cm000
`
`80000
`00<95
`
`00<0<<<00
`
`0000.0003
`
`w...203<0000000<
`
`000
`
`8.
`
`00.0.00<
`8..08
`
`30.80
`.0020
`
`Q00<<
`00<:00
`0<<0<0
`
`000
`
`0<000.0000.
`
`00<:3K0
`
`:300.0
`
`0<<<00
`000.0<0
`
`0.38.0
`000:00
`
`
`
`000.00.0000.000
`
`
`
`000.:3000000
`
`000
`
`0.000<0
`
`000:00
`
`<00<<<
`
`0000000
`
`
`
`
`
`T000.000.00<00<
`
`2000000<0o:
`as.3as0008:
`
`00.<00.0<0000.<
`
`00<0<000.0<0<
`
`0.00.00<=300<
`
`00.<00<
`
`0:0000.
`
`00m
`
`805.00000800...
`
`
`
`20.00....3.0000.0
`
`00.00.00000
`
`E2.020
`
`<00.
`
`.80
`
`00.<
`
`00:
`
`0.0.<
`
`0:
`
`00.0<0<000.
`
`:300<.000.
`
`<00.
`
`:3
`
`00.<
`
`0:
`
`0<0.0<<000.0.00
`
`0<0
`
`m0:
`
`8.0
`
`
`
`Am:.000
`
`000
`
`80
`
`00.0
`
`:3
`
`0.0
`
`.00
`
`0<0
`
`0000
`
`8.0
`
`0a
`
`00<
`
`0.00.
`
`0<<
`
`0.0.0
`
`0<<
`
`0.00
`
`000.
`
`000
`
`08
`
`0s
`
`00000.0
`:3:0020
`
`:00
`
`00.00<0<000<0
`
`0<00.00.<0.0
`
`m0:00000>
`
`00<0<0.000<00
`
`.00.000.02..
`
`0000000<<00<
`
`8000<00.0002
`
`0<0
`
`00<
`
`0<0.
`
`000.
`
`000
`
`00<
`
`00<
`
`0.00.
`
`2
`
`
`
`
`
`
`US. Patent
`
`Nov. 12, 1996
`
`Sheet 2 of 21
`
`5,574,136
`
`3..
`
`0.0.0.
`
`.50
`
`<0.0
`
`0.000
`
`.55.0
`
`.5
`
`2..
`
`.0.<0
`
`m0:
`
`000
`
`m0<
`
`.0.<0
`
`m000
`
`0.0.0.
`
`200
`
`<00.
`
`Em
`
`.5
`
`2...
`
`0.0.0.
`
`205.
`
`00.0.
`
`.50
`
`000
`
`8.0
`
`0<<
`
`m3
`
`000.
`
`00.0.
`
`0.0.<5E
`
`20
`
`00.0.
`
`.000.
`
`.E50
`
`0<0
`
`£00
`
`<.0.<
`
`000
`
`500
`
`0<<
`
`90.0
`
`0.00
`
`..2
`
`<5
`
`5.0
`
`0.0.0.
`
`20.0
`
`5....
`
`.2.
`
`0<<
`
`.5<
`
`0.0.0.
`
`20.0
`
`00.0
`
`055.5
`
`000
`
`0.0.<00<
`
`200.0.0.
`
`.5<000
`
`amm0<
`
`000m
`
`.2..5E05
`
`00.0E0....
`
`000
`
`20
`
`0.0.0.
`
`pom
`
`0000
`
`.80
`
`0.0.0.
`
`
`
`00.0..5<
`
`0<<
`
`o...
`
`2...
`
`00<
`
`.Sm
`
`000
`
`2.0
`
`0.0.0.
`
`.50
`
`US
`
`.2...
`
`5...
`
`a...
`
`00<
`
`50
`
`0.0.0.
`
`.80
`
`55U55...5I5
`
`.52...52...
`
`:00
`.0.<<
`
`000
`
`0.0.<500<000
`<00‘<<<
`.5.5Q5.5
`2.....58..o...
`u0<$0.0
`000<<0.0.<0
`0<<0<0.000m5:5W000
`.5..25E.5.5
`
`.5<0.00.:2
`03.000000
`
`.0.<.0.00<
`0<<.0.<<0<0.
`
`.E...
`
`0.0.0<0<
`05>0.0.0.
`2..25E5..a...
`
`000.
`
`.0.<0
`
`.5<
`
`0.0.0
`
`0m>
`
`000
`
`52
`
`00<
`
`.80
`
`8..Us
`
`m...a.
`5002.0<0<
`
`<00.50
`
`.0.<0
`
`m000
`
`0.00
`
`80
`
`90.0
`
`5...
`
`000.
`
`000.50
`
`208.0
`
`0<0
`
`=00
`
`0<000<
`
`
`
`m000:00.
`
`0<00.0.0
`
`
`
`H.00.50
`
`$00
`
`<00.0.<0
`
`2.0.5<
`
`5...5..
`
`0<00.0.0.5m...
`
`:00m000.0
`
`.5.2<580
`
`000
`
`00<
`
`0.0.0.
`
`.0.<0
`
`m0:
`
`0.0.0
`
`05.0
`
`.5
`
`m...
`
`000<.0.<
`2020
`.2.5a:5..
`
`0000
`
`0<<00.0
`
`0.0.00.0.0.
`
`m3:3
`
`0.00200
`
`.5
`
`8.
`
`00<
`
`0.0.0.
`
`5..
`
`.2.
`
`0.0.000<
`
`
`
`H5.0:00.
`
`comm
`
`000
`
`2.0
`
`0<0
`
`:00
`
`0.0.0.
`
`.50
`
`000.
`
`“20.0
`
`.50
`
`8.0
`
`.5<00.<
`
`.50030
`
`000.52
`
`8.0.5<
`
`0.00<0<
`
`m0<00<
`
`
`
`<0000.0..0.<<00.0.
`
`2.0$0.5<.020.
`
`00.0.
`
`.50
`
`80.0
`
`0<0
`
`:00
`
`.5
`
`a...
`
`000
`
`0000
`
`030H00<£0050.0
`
`
`
`0.0.<000.0.<00.0.0
`
`<00
`
`00<
`
`00<
`
`<<00<0<0<0.0.<0.0.<
`
`
`0000.000.000.000000.0.00<0.0.0
`
`000
`
`0.0.0.
`
`H.00:000.0.0.0e:«.00
`
`
`8.0:3.000..80Sm
`
`0<<
`
`00.0.0<000<000<<<
`
`0<0<<0.0<00.5<<0
`
`a.52.
`802.0%.5.80E
`.000.E:0.00.8.090.0
`
` .055....5000%m<:00:00:3=00
`2..2...25.55..5..
`
`<<0
`
`:00
`
`90.0
`
`2..
`
`.5<
`
`.50.
`
`0.5
`
`.50
`
`u...
`
`a...
`
`00<
`
`.50
`
`0.0.<
`
`m.00
`
`0.0.0
`
`05>
`
`a....5
`
`2...3.
`
`5....5
`
`2mm...
`
`a..5.58.
`
`.00.5E8..
`
`m.00
`
`.500000<0.50<00
`
`80<0<
`:008005>
`
`000
`
`E:
`
`
`
`0.500.0.0.0.<0<<<5
`
`0<0
`
`:00
`
`.5
`
`a.
`
`0.0.0
`
`0m>
`
`00.0.
`
`.80
`
`E......200<00.0.
`
`5.5
`
`<00<00
`
`a2.05
`
`0000
`
`.5<
`
`Sm
`
`00000
`
`20.0now
`
`000m
`
`.50
`
`o...8..E2...
`
`.52...2...5.2.
`E.2..2..5<55..so5..
`
`m0<
`
`.5<
`
`.3.5
`
`0<0
`
`:00
`
`000
`
`0.0.0
`
`030
`
`0<0
`
`30
`
`0<<
`
`0<0
`
`.5<
`
`8...
`
`.005.5
`20:00
`
`0.0.<55..
`
`.8020:3:320.0
`900new8.0
`
`M.00
`
`MW0<
`
`0.0.<
`
`500
`
`2..5.5.5
`
`:00
`
`3.020.005>
`
`5..E5.5a...E5...5
`
`on...
`
`.50
`
`8.0
`
`00.00.500<
`
`3.03.0Sm
`
`0.000
`
`000.0
`
`.238.0:30<<
`<00.05PE
`
`20
`
`55..
`
`....2..55...5..
`
`.5.85....<5o...
`
`
`0.0.00.0.0.0.0.0
`
`0<0
`
`<00.0000.0.00.50.0.0.
`0.0.0.00<0.0.0
`
`<<<00.0.00.0.0500.0.0
`
`«.00500.050000.0.“m0“00>00>”.0000.0.”EHEm
`
`
`0<.0.0<50.0.0.00<.5<00.0.
`
`
`5.E
`
`00.0.0.5<0.<000
`
`v.00
`
`a...2.15
`
`000
`
`a0<
`
`0.0.<
`
`0<0
`
`:00
`
`2..
`
`5..
`
`0<0<0005
`
`00.0
`
`8.0.5<2.0030
`
`<00.<000.0.000<
`
`.808.00....8m
`
`0.000
`
`.5.58..5..
`
`.2a..52..
`
`.0.<0
`
`.5<
`
`00.0.
`
`.50
`
`0.0.0
`
`50
`
`0<0
`
`.5<
`
`0.0.<
`
`0o:
`
`.50....
`
`0.0.0
`
`05>
`
`.2.5
`
`0.0.0
`
`0m>
`
`0.0.0
`
`.50
`
`0<0.0000<00.0.<0<0
`.0..2.55..0....5m
`5..0:
`
`8..2...5a....88..
`
`5..
`
`2..
`
`000.0
`
`<0<.5<
`
`08
`
`00<00<0<<
`<00.3500.0.
`
`
`
`:00.0.0.0.
`
`2..
`
`Em
`
`0<0
`
`:00
`
`0<0
`
`0<0
`
`50
`
`0.0.0
`
`05>
`
`000.
`
`000
`
`2.0
`
`0<<
`
`.5<
`
`000.
`
`0.0.0
`
`0.00
`
`0<0<00
`00.0.00<<0<
`
`:00«E
`
`.0.20.0.0.00.
`
`
`00.0.00.0.
`0<00<0000
`
`000050
`
`00<
`
`0.00.
`
`000
`
`52
`
`000
`
`0.0.<
`
`0000
`
`:30.0.0
`
`0<0.
`
`0.0.0.
`
`.5<.000
`
`m0:200
`
`000<<0
`
`H.0<
`
`:00
`
`.000
`
`80
`
`<<0
`
`=00
`
`0.0.<
`
`m.00
`
`55..
`
`.E50
`
`<0.<
`
`20
`
`<00
`
`20
`
`.5055..
`
`3
`
`
`
`
`
`
`US. Patent
`
`Nov. 12, 1996
`
`Sheet 3 of 21
`
`5,574,136
`
`55.3EasE
`0.880
`:3Em
`
`:5
`
`comm5anasE3Us
`3:.<8
`E.Rm
`
`Em
`
`was“$2.
`235EE
`
`8mm
`
`on“.
`
`commoovm
`
`3o.5.8h.03.5“.08.5.us.go8%<5<8<809.8<802%25E95cg.50.5.v.8Bu
`
`o:EEE.5EEa:5.5E.E55EEEam:5.3E55E.3E
`
`
`
`
`
`Eu<80895.E.0%Eu.068o<3<8..58“E.8...o:3256.5.E..3..Eu0%.50on:08
`
`3:.UE
`
`2mas2m3»BE.5.Eu:52ma:SmmiofAE.my=:3.Eu.5.8m93PE.mm2mEh.8;
`
`ENE
`
`cow5.5=5:5E5EE5=35.52::3EasE.2:EE52aEasE80.335E«8EEasEasasEEEEEEEEasEEEUs
`363.588oSEuewwwqueufiuuu3453ufio<u<u$8uu$<§<<<u$uoowmwwu<ufiu<uu§<ut<$ouchuéfiuEEEEoEowe
`
`fiEfisfianSoESudoewuyfiuuouéfiwwufifiéfigéfiwufieufififififi<804.95298uowmmwuuufisgfiufififiufiufig
`
`Son
`
`EmuE<wmwwu<uu§ufiuEsaubfiufiouaégfiu2.5ficuEuEfiuuSuuSEEufiEpfiuuEuuquw<wEucautgwmwwctfiobu
`
`EuEEEflu38EufiwgwwwwwofiufioBfiEmowiuwSuSpageEBSEEQSefiuuiggufifi«Sous—buuSuufiuuufioEu
`
`88
`
`USSEEEEEQESugoufifiécgpufiuoggggfis33898¢u“V5obuéfimwmmfiguufiogouu38E.
`
`4
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 4 of 21
`
`5,574,136
`
`dd002
`
`IOOC
`
`IOOC
`IeS
`
`III
`IIIPUIH
`
`2. I fºd
`
`29Id
`
`5
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 5 of 21
`
`5,574,136
`
`
`
`
`
`O
`
`1500
`1 000
`500
`G-CSF added (pM)
`FIG 3A
`
`0.3
`
`0.2
`
`s
`
`0.1
`
`0.0
`
`0.3
`
`0.2
`
`s
`
`a
`
`0.1
`
`O
`
`20
`40
`Bound (pM)
`
`80
`
`O
`
`1000
`500
`1500
`G-CSF added (pl.)
`FIG 3C
`
`0.0
`
`O
`
`60
`
`40
`20
`Bound (pM)
`FIG. 3D
`
`6
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 6 of 21
`
`5,574,136
`
`120
`
`
`
`
`
`(?oinuoo Jo Z) punoq JSO-?–
`
`I
`9
`2
`|
`
`
`
`(OH,O) JSO-? nH
`
`
`
`(1100'E) JSO-?) nH
`
`Competitor
`
`FIG.
`
`4
`
`7
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 7 of 21
`
`5,574,136
`
`COS(plb2)
`*
`*
`-
`w
`
`x
`
`NFS60
`
`.
`
`.
`
`DSS DST
`cold G-CSF
`&:
`
`Ori.
`
`(kDa)
`2OO .
`
`
`
`
`
`
`
`8
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 8 of 21
`
`5,574,136
`
`S one
`
`8 S more
`
`
`
`FIG.
`
`6
`
`9
`
`
`
`US. Patent
`
`Nov. 12, 1996
`
`Sheet 9 of 21
`
`5,574,136
`
`-Eu
`
`31040
`
`m e >
`UCuZ
`
`,---
`IJIQ 2-;
`."""
`,-.J
`€3.93"
`mica:
`
`FIG.7(a)
`
`
`
` 2.: L.J::L.J
`
`VNN
`r-H‘Ch
`V‘VC‘
`mOv—l
`NHr—i
`NNN
`
`:1:
`m
`Lu
`Lu
`w mm m
`mcfi
`quc UACCC
`[04.30
`”14:0
`(90:00 (90100
`
`10
`
`cons
`
`
`RPLR GHR
`
`10
`
`
`
`Sheet 10 of 21
`
`5,574,136
`
`US. Patent
`
`ch
`AU)
`Om
`
`5:6}
`
`NOV.12,1996
`r---1
`IAI):
`L..-_.J
`IIQ4
`M l
`r—-..1
`I L-‘l>'
`L~-_J
`>Cd
`hi4
`C)?‘
`
`Faxr~‘-n
`IV] {9'
`~-_-J
`PI?‘
`Ztn
`
`FIG.7(b)
`
`EFQLHNTRY
`
`1 I I I J
`N K
`i L
`iN
`l I
`r-
`L-
`
`)
`
`G-CSFR(470)
`
`CONTAC(840)
`
`AH
`mo
`
`G—CSFR(51
`
`CONTAC(89
`
`G—CSFR(569)
`
`CONTAC(938)
`
`11
`
`W W
`
`R'Y
`E_
`I I
`L Q
`
`._fvgcy
`
`'I I I J
`{E 1§
`LL EKI I I I
`r I I I L
`
`J?
`
`T T
`
`r-—-¢:__P____:E‘SL
`Tgss 03;?
`szfilSl<Gv
`
`N N
`y. E
`IA
`
`WEAP WH}{V
`
`D S
`V V
`W S
`
`EI
`
`T E
`
`'WU')‘
`
`G-CSFR(37
`
`CONTAC(74
`
`~CSFR(422)ONTAC(795)
`
`G C
`
`11
`
`
`
`US. Patent
`
`Nov. 12, 1996
`
`Sheet 11 0f 21
`
`5,574,136
`
`r'--1
`10:2. L“:
`mm} 3ch
`Era {+533
`mo
`:20
`02—1
`{AA
`
`351:9} >«u
`0s: mm
`
`{is Mm
`89.63
`rm:
`HI mm m:
`can
`aux mm
`
`iE—Ta‘fi >0
`
`0:1
`
`{5413.
`
`0.4
`
`29.9
`00 {L259} >0
`on: A2
`ore—I
`
`vfi‘:GLIJ PL PL ."G'EL5L
`
`L:
`
`0””
`>_«_z
`91".”
`flu >4";
`{3:93 {5—1 a}!
`921
`Gm
`n>AIIA IH
`l'""1 1""!
`'
`'
`cm: .mo-
`3“ ‘0: L37;
`U):
`a:
`“31::
`C“
`'s----1
`.--—-1
`04.0
`595.} $52
`0:4 -L cu] mo
`§5>4
`C35*
`iiii
`[:4
`mo
`- 22.. u
`HO KGB-g PIA: >O4
`mo mu W
`mo-
`'05:;
`om
`,9z
`.LIO;
`
`I
`I
`I
`
`‘
`.
`m
`«I
`u»
`a
`
`I
`|
`
`'
`
`was
`>e«
`mo
`
`Om :z—Im:
`
`’
`
`‘
`
`0
`‘-—’
`p
`
`'
`(5
`I-—I
`
`r“"\
`1615;
`34>
`m
`3 I
`WI
`
`w
`
`mm;
`
`mm:
`
`H
`
`cm
`m
`PH:
`r a l I L
`:oo<
`s o
`
`L‘i‘..J
`u:
`I
`6* I
`XH
`r"'
`ox”
`0‘
`
`
`
`r---~
`'-~-
`r---1
`9‘3
`>+C3
`'DJCg
`fill
`Md: Ad Am fig
`
`Nth Nm
`0O LOU)
`Ian: mm
`vv vv
`
`Chv
`Nt‘ Nv‘
`(‘1!) No min
`\OU')
`l‘kD
`hm
`VV vv vv
`
`a:
`at
`m
`m
`a:
`Luci
`[no.1
`Luci
`Luca
`Luci
`cav-
`(nv mv mv
`mv
`()1
`Ul U!
`U! U!
`IA IA
`IA.
`IA IA
`0H (DH
`L’JI—I OH OH
`
`12
`
`12
`
`
`
`U.S. Patent
`US. Patent
`
`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 12 of 21
`Sheet 12 of 21
`
`5,574,136
`5,574,136
`
`1N
`C
`N-1
`N
`\
`
`dv 1
`
`A "
`
`CD
`(.5
`m
`I—a
`t
`LT-u
`
`d
`o
`
`s
`S
`H
`E-'
`
`13
`
`CN
`va
`O
`
`mH m
`
`as
`S
`O
`no
`
`g
`g
`Q
`0’3
`
`317
`
`95
`
`13
`
`
`
`U.S. Patent
`US. Patent
`
`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 13 of 21
`Sheet 13 of 21
`
`5,574,136
`5,574,136
`
`
`
`ésgueuooogwégashgsfiggbgggaggiggcuccgansgéaggg<
`
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEESEEEEME
`
`so8;E2:3Eng:65Eo:5Em5E:EE35:39:_°>0:2;ca3E5Ew.__._5Eu:5=5_05803055038b”3”33o:0:8h088a<202805usso3a05o:088o<8058‘a:068o08053an
`
`ICIEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEm
`
`“@EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEa:
`
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
`
`EsEEEEEEEEEEEEEEEEEEEEEESEEEEEESEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEm
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
`
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE_EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
`
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEa
`
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
`
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
`
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE:
`
`EEEEEEEEEEEEEEEEE;,EEEEEEEEEEEEEEEEEE
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE_E
`
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE2
`
`EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
`
`W
`
`3m.wE
`(e)g '?IJ
`
`
`14
`
`14
`
`
`
`
`U.S. Patent
`US. Patent
`
`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 14 of 21
`Sheet 14 Of 21
`
`5,574,136
`5,574,136
`
`
`
`§E§E§EE§EEEEEEE§E§§E3§§EEE§§§§§E§EE
`
`afiéfiésifiésaaéEiéééE=55§§§é555§5555§
`
`E§§EE§§§E§EEEEEE§§§E§§EESSEE§EEEE§§
`
`Esgiaaségaafisfiéiééaéfifiéfiésasfiéaééié
`
`§§§33§5§§§§§§E§§EEEE§§§§§S§§§§§E§E§
`
`EEEE§£§££$££5§5£53355££E£§££§£53§§§
`
`
`
`
`
`ESSHUEBuSuBHEBuUBEUBEUu2u5538329t8<8<3:592E§8<u<<u8§u<u§o8u<u§
`
`
`
`
`
`
`
`
`
`9:.ESEa:a:smegmsmsueasu55535£55.£2.232;an=353552m=55
`
`{J
`e
`
`§EE§E=Esfl§§§E§§§EEE§EEEE§§§EE§EEEE5
`
`e
`
`
`
`ofiUBWEUEFEEuEuBBZSEEUE8<EEEEE§92.2.3chEgoaguéuauccumvauugg
`>-駧£§=fiagé£5£5§££§§§aééiiaééiaéiaéa
`
`=3amamo=3=2E.ao=3.5.=3=3=3=3=3823=35=39:u:3:=3=5.3.5=52;.5.=3
`
`.3EuE
`
`E8a98caHE.8a2%”.Eu2....p:<2=895we.8.—95us840295.5<8as<895k:<82:.958a.3a:84
`
`
`
`
`
` .0E53£3555:=E£2£$5§£$飣§é§£££§£§3§
`
`3m.wE
`
`.3a55EE3an..=.EEaE5E.5.5..=.5.3EaE5..=aEEE5E3%EE§§E§§35§E§§E§EESEEEEE§EE§§§§§§
`
`§§E3%EEE§E§§§EE§§§§§§E§§§E§§EEE§EEE
`
`gaifiéasfi35535535355553333535553555E
`
`éE§EEEE§E§§§§E§§EE§§§§EE§EE§§§§§§§§
`
`éésifiéb駣£éé§5£§§£5E§E§E§E飣5££éé
`
`EE§E§EEE§EEEE§§EEEEE§E§§§§§EE§§E§§§
`
`£33aaéifiééfiiiéaéagafiéégéfiééEfiésfiééfi
`
`oEuEuEEbouEfiosasu"SEES$8328EEEEEEBEBEucu2.3842853upspssopgaufiuguapgausgg
`
`Em2:am5=3=2=6.3—5582884833538583205838865488382308358UESSEEBESEa:9:.a:8a958oeaa:as
`
`OBSEEEEE
`
`8:
`
`E.
`
`32
`
`8..
`
`5v8:
`
`:2
`
`an
`
`23
`
`an
`
`$8
`
`an
`
`$3
`
`38
`
`999
`=3
`
`$3
`
`2:.
`
`mam
`
`E.
`
`#998
`.33
`
`EN
`
`3mm
`
`15
`
`15
`
`
`
`
`U.S. Patent
`US. Patent
`
`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 15 of 21
`Sheet 15 of 21
`
`5,574,136
`5,574,136
`
`onw.wE
`(0)9 ':)IJ
`
`
`QEééfi§3§§§§§§§§§E§§3§33§§§E§§§§%§§E
`EEEEEEEEEEEEEEEEEEEEEEEEEEo:EEEEEEEE
`
`so<23so<2<280Q8Q5Q02580%«2<2a:028‘.020205<2“Q5<2<805085a:So80080%02
`E«E5c2.5a:2;mm52;as3am3m:E.E3“E«55amam2;ama;5maca:3ca5ma5am
`
`“a:2:am30:c22;c2:3EE52;CE25555E5oz5m5z:e;a2.8HS=32;am5Ea:Eas5%Q:5<8so29Q8038o0%08<8Eas5<50%805%R:8*Q:so5202om:0:<80885as
`
`E.2;am21c2en.955oz3BEam2;am:3DEEEa:5a:E5555c255c2a:a”:3am5”EbeE«8so<880<2855802E8002”E«8Q.05E02”E:208050855853<8<80202020%08
`
`EEE5a;2;ca5oz55am5S85588EEG§8§5§838§88<55880<8R:E08<88‘.on:55§8<<3
`
`8n
`
`mow
`
`ia:
`
`m5
`
`EN
`
`§32
`
`on
`
`5%
`
`16
`
`E535EEEEEEEEEEEEEEEEEEEEEEEEEEEEE
`EEEEEEESEEEQEEEE§§§§E§§§§§§§§E35E
`
`999
`100%
`m8
`5%
`
`16
`
`
`
`U.S. Patent
`US. Patent
`
`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 16 of 21
`Sheet 16 of 21
`
`5,574,136
`5,574,136
`
`O)
`
`9 F
`
`IG.
`
`VIZ/III/I/A
`
`
`
`
`
`pHQ2
`
`pHGl1
`
`17
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 17 of 21
`
`5,574,136
`
`
`
`
`
`O
`w
`
`0.
`CD
`t
`
`m
`
`<g
`O
`vm
`
`CD
`H
`r
`
`eed/puno
`
`s
`
`r
`g
`
`O
`O
`O
`Q
`
`e 3
`S
`
`g
`s &
`th
`l
`
`vu
`
`O
`
`18
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 18 of 21
`
`5,574,136
`
`(várod), een
`
`ueror), con
`
`? ººx
`
`º 9 **+++
`
`equa o end
`
`<,
`
`() u.c.) eru a oeddD
`
`
`
`19
`
`
`
`U.S. Patent
`US. Patent
`
`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 19 of 21
`Sheet 19 of 21
`
`5,574,136
`5,574,136
`
`
`
`
`
`
`
`20
`
`20
`
`
`
`U.S. Patent
`US. Patent
`
`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 20 of 21
`Sheet 20 of 21
`
`5,574,136
`5,574,136
`
`
`
`
`
`
`
`21
`
`21
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 21 of 21
`
`5,574,136
`
`
`
`OOC
`
`N
`
`III PUIH
`
`III PIH
`
`22
`
`
`
`5,574,136
`
`1.
`DNA ENCOOING GRANULOCYTE
`COLONY-STMULATING FACTOR
`RECEPTOR AND PROTEIN THEREOF
`
`FIELD OF THE INVENTION
`This invention relates to an isolated DNA encoding granu
`locyte colony-stimulating factor receptor. More particularly,
`it relates to an isolated DNA encoding a receptor peptide
`capable of specifically binding to a colony-stimulating factor
`(hereinafter, referred to as G-CSF), an expression vector
`containing said DNA, a transformant transformed by said
`vector, and a process for the production of said receptor by
`culturing said transformant. The present invention also
`relates to a recombinant G-CSF receptor prepared according
`to the present process.
`
`10
`
`5
`
`2
`Leukemia, 1, 1-8 (1987); and Park et al., Blood 74, 56-65
`(1989)). Human G-CSF is a 174 amino acid polypeptide
`while murine G-CSF consists of 178 amino acids. Human
`and mouse G-CSFs are highly homologous (72.6%) at the
`amino acid sequence level, in agreement with the lack of
`species-specificity between them (Nicola et al, Nature 314,
`626-628 (1985)). What makes the research in G-CSF more
`interesting is that G-CSF receptor has also recently been
`found in non hemopoietic cells such as human endothelial
`cells (Bussolino et al., Nature 337, 471-473 (1989)) and
`placenta (Uzumaki et al., Proc. Natl. Acad. Sci. USA, 86,
`9323-9326 (1989)).
`As can be seen from the above, the elucidation of the
`interaction between G-CSF and its receptor should greatly
`contribute to the development of the treatment or prophy
`laxis of various diseases including hematopoietic disorders
`using G-CSF, whereby providing more effective and proper
`treatments on such diseases. Thus, such elucidation is impor
`tant not only academically but also clinically. On the other
`hand, the receptor itself can be useful. For instance, a soluble
`form of the G-CSF receptor may be useful clinically to
`inhibit the proliferation of some G-CSF-dependent human
`myeloid leukemia cells (Santoli et al., J.Immunol. 139,
`3348-3354 (1987)). The investigation into the expression of
`G-CSF receptor in tumor cells such as myeloid leukemia
`may be beneficial to establish an effective clinical applica
`tion of G-CSF. Accordingly, owing to the various academic
`and practical usefulness, a stable supply of a G-CSF recep
`tor-encoding gene and the G-CSF receptor has been
`demanded.
`Recently, the technology of genetic engineering has been
`used for the production of various physiologically active
`Substances. The production by the genetic engineering is
`generally carried out by cloning DNA encoding desired
`polypeptide, inserting said DNA into a suitable expression
`vector, transforming an appropriate host cell such as micro
`organism or animal cell by the expression vector, and
`making the transformant express the desired polypeptide.
`To apply the genetic engineering technique to the pro
`duction of G-CSF receptor, cloning of DNA encoding
`G-CSF receptor is firstly required. However, cloning cDNA
`encoding G-CSF receptor was hampered by the low number
`of receptors present on the cell surface (at most hundreds to
`2,000 receptor per cell).
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 (A-C) depicts the nucleotide sequence and deduced
`amino acid sequence of murine G-CSF receptor (SEQ ID
`NOS:1-2).
`FIG. 2(A-C) depicts a schematic representation and
`restriction map of murine G-CSF receptor cDNAs (p162,
`p17 and pFl), and hydropathy plots thereof.
`FIG. 3a depicts the saturation binding of murine 'I-G-
`CSF to COS cells.
`FIG. 3b depicts scatchard plot of binding data of murine
`'I-G-CSF to COS cells.
`FIG, 3c depicts saturation binding of murine 'I-G-CSF
`to NFS-60 cells.
`FIG. 3d depicts scatchard plot of binding data of murine
`'I-G-CSF to NFS-60 cells.
`FIG. 4 depicts specific binding of murine G-CSF to
`recombinant murine G-CSF receptor expressed by COS
`cells.
`FIG. 5 depicts crosslinking of murine G-CSF receptor
`expressed in COS cells and those expressed by NFS-60 cells
`with
`I-G-CSF.
`
`30
`
`35
`
`BACKGROUND OF THE INVENTION
`Proliferation and differentiation of hematopoietic cells are
`regulated by hormone-like growth and differentiation factors
`designated as colony-stimulating factors (CSF) (Metcalf, D.
`Nature 339, 27-30 (1989)). CSF can be classified into
`several factors according to the stage of the hematopoietic
`cells to be stimulated and the surrounding conditions as
`25
`follows: granulocyte colony-stimulation factor (G-CSF),
`granulocyte-macrophage colony-stimulation factor (GM
`CSF), macrophage colony-stimulation factor (M-CSF), and
`interleukin 3 (IL-3). G-CSF participates greatly in the dif
`ferentiation and growth of neutrophilic granulocytes and
`plays an important role in the regulation or blood levels of
`neutrophils and the activation of mature neutrophils
`(Nagata, S., "Handbook of Experimental Pharmacology',
`volume "Peptide Growth Factors and Their Receptors', eds.
`Sporn, M. B. and Roberts, A. B., Spring-Verlag, Heidelberg,
`Vol.95/1, pp.699-722 (1990); Nicola, N. A. et al.,
`Annu.Rev. Biochem. 58, pp.45-77 (1989)). Thus, G-CSF
`stimulates the growth and differentiation of neutrophilic
`granulocytes through the interaction between cell-surface
`receptors on precursors of neutrophilic granulocytes to give
`40
`mainly the neutrophilic granulocytes (Nicola, N. A. &
`Metcalf, D., Proc. Natl. Acad, Sci. USA, 81, 3765-3769
`(1984)).
`G-CSF has various biological activities in addition to
`those mentioned above. For example, G-CSF prepared by
`recombinant DNA technology has proven to be a potent
`regulator of neutrophils in vivo using animal model systems
`(Tsuchiya et al., EMBO.J. 6 611-616 (1987); and Nicola et
`al., Annu. Rev. Biochem. 58, 45-77 (1989)). Recent clinical
`trials in patients suffering from a variety of hemopoietic
`disorders have shown that the administration of G-CSF is
`beneficial in chemotherapy and bone marrow transplantation
`therapy (Morstyn et al., Trends Pharmacol. Sci. 10, 154-159
`(1989)). It is also reported that G-CSF stimulates the growth
`of tumor cells such as myeloid leukemia cells.
`55
`Despite the biological and clinical importance of G-CSF,
`little is known about the mechanism through which G-CSF
`exerts its effects. Therefore, it has been needed to elucidate
`such mechanism to establish more effective treatment and
`diagnosis for G-CSF-related disorders. For this purpose, the
`biochemical characterization of specific cell-surface recep
`tors for G-CSF and the evaluation of interaction between
`G-CSF and the receptor must be performed.
`Several reports suggested that the target cells of G-CSF is
`restricted to progenitor and mature neutrophils and various
`myeloid leukemia cells (Nicola and Metcalf, Proc. Natl.
`Acad. Sci. USA, 81, 3765-3769 (1984); Begley et al.,
`
`45
`
`50
`
`60
`
`65
`
`23
`
`
`
`5,574,136
`
`3
`FIG. 6 depicts northern hybridization analysis of murine
`G-CSF receptor mRNA.
`FIG.7(A-D) depicts alignment of amino acid sequence of
`murine G-CSF receptor and those of other growth factors
`and schematic representation of murine G-CSF receptor.
`FIG. 8a depicts the nucleotide sequence and deduced
`amino acid sequence of plasmids pHQ3 and pHG12 (SEQ
`ID NOS:3-4).
`FIG. 8b depicts the nucleotide sequence and deduced
`amino acid sequence in plasmid pHQ2, said sequence being
`different from that of pHQ3 and corresponding to the
`sequence downstream from nucleotide 2,034 in said pHQ3
`(SEQ ID NOS:5-6).
`FIG. 8c depicts the nucleotide sequence and deduced
`amino acid sequence of the insertion present in pHG11, said
`sequence being inserted in pHQ3 (SEQ ID NOS:7-8).
`FIG. 9 depicts a schematic representation and restriction
`map of pHQ3, pHG12, pHQ2 and pHG 11 described in FIG.
`8.
`FIG. 10a depicts saturation binding of murine I-CGF to
`COS cells.
`FIG. 10b depicts scatchard plot of G-CSF binding data.
`FIG. 11 depicts northern hydridization analysis of human
`G-CSF receptor mRNA.
`FIG. 12 depicts detection of human G-CSF receptor
`mRNA by PCR.
`FIG. 13 depicts southern hybridization analysis of human
`G-CSF receptor gene.
`FIG. 14 depicts schematic representation map of expres
`sion vector pEF-BOS.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`4.
`properties commonly found in these members (FIG. 7). A
`probe prepared from thus obtained murine G-CSF receptor
`DNA hybridized with human G-CSF receptor, demonstrat
`ing that human and murine G-CSF receptors are highly
`homologous. Therefore, murine G-CSF receptor can serve
`as so-called "an intermediate' for the preparation of human
`G-CSF receptor.
`As the next step, the present inventors studied with a
`purpose of providing sufficient amount of human G-CSF
`receptor and succeeded in the isolation and cloning of cDNA
`encoding human G-CSF receptor by isolating total mRNA
`from human placenta and U937 cells, constructing cDNA
`library, and screening said library using a probe prepared
`from murine G-CSF receptor cDNA.
`When COS cells were transfected with a plasmid encod
`ing human G-CSF receptor of the present invention, said
`cells expressed a receptor which has similar specific binding
`properties to that of the native human G-CSF receptor.
`Accordingly, the present invention provides an isolated
`DNA encoding G-CSF receptor. The present invention fur
`ther provides an expression vector containing G-CSF recep
`tor DNA. The present invention also provides a method for
`producing G-CSF receptor which comprises transforming a
`host cell by the expression vector, growing said transformant
`in a medium, and recovering G-CSF receptor.
`For purposes of the invention, the term "G-CSF receptor
`peptide' herein used refers to both the mature G-CSF
`receptor peptide and peptide fragments thereof, said frag
`ments having an ability to bind specifically to G-CSF.
`The human G-CSF receptor, which has been hardly
`obtained heretofore, can be easily prepared by the genetic
`engineering by virtue of the present invention, and in turn
`used for many studies directed to, for example, the eluci
`dation of the mechanism through which the G-CSF and/or
`G-CSF receptor exerts the effect, the clinical (for diagnosis
`and treatment) application, as well as for the promotion of
`the practical application. Additionally, probes obtained from
`G-CSF receptor cDNA can facilitate the detection of G-CSF
`receptors on tumor cells such as leukemia cells before the
`clinical application of G-CSF during the treatment of
`patients suffering from these diseases. Therefore, said cDNA
`is useful to perform an effective clinical application of
`G-CSF. Furthermore, proteins or compounds capable of
`binding to G-CSF receptors can be developed by investi
`gating into tertiary structure of soluble-form G-CSF receptor
`which are prepared in large scale by the DNA recombinant
`technology.
`Cloning of DNA encoding murine G-CSF receptor was
`carried out as follows. Thus, G-CSF receptor was initially
`purified from mouse myeloid leukemia NFS-60 cells which
`have relatively higher expression of the G-CSF receptor and
`determined the molecular weight of about 100,000 to 130,
`000 dalton. Total RNA was prepared from NFS-60 cells by
`the guanidine isothiocyanate/CsCl method, and poly(A)
`RNA was selected, which was then used for the synthesis of
`double-stranded cDNA using the reverse transcriptase, DNA
`polymerase and the like. A cDNA library was constructed in
`the mammalian expression vector CDM8 (Seed, Nature 329,
`840–842 (9187)), as 884 pools of 60 to 80 clones. Plasmid
`DNAs from each pool were prepared and introduced into
`COS-7 cells. Two positive pools I62 and J17 which showed
`significant binding of radioiodinated G-CSF were selected.
`From these pools were identified plasmids pl62 and p17
`which have higher binding activity with G-CSF. When
`plasmids p162 and p17 were transfected into COS-7 cells,
`resultant cells expressed receptors capable of binding to
`G-CSF.
`
`DISCLOSURE OF THE INVENTION
`The finding that human and mouse G-CSFs are highly
`homologous (72.6%) lacking species specificity lead to an
`presumption that these G-CSFs probably cross-react. Thus,
`present inventors have studied for the purpose of obtaining
`a G-CSF receptor which can be used in the research as well
`as the diagnostic analysis, and succeeded in the purification
`of the receptor as a protein with a molecular weight (M.W.)
`of 100,000 to 130,000 from a solubilized mouse G-CSF
`receptor from mouse myeloid leukemia NFS-60 cells. The
`purification can be carried out by, for example, extracting
`cell membrane suspension with CHPAS (3-(3-chola
`midepropyl)dimethylammonio)-1-propanesulfonic
`acid),
`treating the extract with G-CSF-affinity chromatography
`prepared by binding recombinant human G-CSF to the gel
`resin, and purifying by gel filtration.
`The present inventors have also succeeded in the isolation
`and cloning of cDNA encoding murine G-CSF receptor
`(hereinafter, referred to as G-CSF receptor cDNA) by the
`reverse transcription of mRNA isolated from NFS-60 cells
`for the first time. Thereafter, the nucleotide sequence of
`cDNA and deduced amino acid sequence were determined.
`The nucleotide sequence and predicted amino acid sequence
`of murine G-CSF receptor cDNA are shown in FIG. 1, and
`are identified in the Sequence Listing as SEQID NO:1 and
`SEQ ID NO:2. When COS cells were transfected with an
`expression cloning vector containing murine G-CSF recep
`tor cDNA, said cells expressed a receptor which has similar
`properties to that of the native G-CSF receptor on NFS-60
`cells. Comparison of the amino acid sequence predicted
`from murine G-CSF receptor cDNA with those of other
`members belonging to growth factor receptor family
`revealed that the murine G-CSF receptor possesses many
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`24
`
`
`
`5,574,136
`
`10
`
`15
`
`20
`
`30
`
`35
`
`25
`
`5
`The determination of nucleotide sequences of resultant
`plasmids p17 and pl62 revealed that the two cDNAs
`contain the complete coding sequence of G-CSF receptor
`but lack the poly(A) tract and poly(A) additional signal. The
`cDNA library was, therefore, rescreened by colony hybrid
`ization using the 2.5 kb HindIII-Xbal fragment of p17 as a
`probe. Among positive clones was selected pF1 which had
`603 bp 3' non-coding region and contained two overlapping
`poly(A) addition signals. The composite nucleotide
`sequence of the three cloned cDNAs (pl62, p.17 and pF1)
`is presented in FIG. 1 together with the predicted amino acid
`sequence, also identified in the Sequence Listing as SEQID
`NO:1 and SEQID NO:2. The schematic representation and
`restriction map of the hydropathy plot of them are presented
`in FIG. 2.
`The murine G-CSF receptor cDNA cloned by the present
`invention has the following characteristics.
`There is a long open reading frame starting from the
`initiation codon ATG at nucleotide position 180-182 and
`ending at the termination codon TAG at position 2691-2693
`(2,511 nucleotides). At the 5' upstream from the long open
`reading frame, three other potential initiation codon ATGs
`can be found at petitions 73, 105 and 126. All of these are
`followed by short open reading frames. Deletion of these
`ATG codon