Note: Descriptions are shown in the official language in which they were submitted.
2 ~
~ e s c r i p t i o n
The invention concerns muteins of the granulocyte
stimulatinq factor G-CSF in the sequence
5~1 52 53 54 55 56
Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 56 of the
mature G-CSF with 174 amino acids or at position 53 to
59 of the mature G-CSF with 177 amino acids or/and at
least one of the 4 His residues at positions 43, 79, 156
and 170 of the mature G-CSF with 174 amino acids or at
positions 46, 82, 159 or 173 of the mature G-CSF with
177 amino acids.
Lymphokines are involved in the maturation of blood
cells. They stimulate the maturation of bone marrow stem
cells to fully differentiated cells. G CSF is
synthesized by activated monocytes, macrophages as well
as by a series of other cell lines.
G-CSF was purified to homogeneity from cell culture
supernatants of the human bladder carcinoma cell line
5637 (Welte et al., Proc. Natl. Acad. Sci 82 (1985),
1526). The sequence of the cDNA coding for native G-CS~'
is known from Sunza et al., Science 232 (1986), 61. As a
consequence of alternative splicing in the second intron
two naturally occurring forms of G-CSF exist with 204 or
207 amino acids of which the first 30 represent a signal
peptide (Lymphokines, IRL Press, Oxford, Washington
D.C., Editors D. ~ale and C. Rickwood). The mature
protein has a molecular weight of ca.l9.6 kD and has 5
cysteine residues which can form intermolecular or
intramolecular disulphide bridges. Binding studies have
shown that G-CSF binds to neutrophilic granulocytes.
None or only slight binding is observed with erythroid,
lymphoid eosinophilic cell lines as well as with
macrophages. The G-CSF receptor consists of a single
peptide chain with a molecular weight of 150 kD (Nicola,
Immunol. Today 8 (1987), 134). The number of receptors
per cell generally increases with the maturation of the
cells~and can amount to several hundred per cell. It is
assumed that lymphokine receptors consist of an
e~tracellular domain, which binds the ligands, a
hydrophobic transmembrane re~ion and an intracellular
domain. Binding of lymphokines to their receptor can
cause the synthesis of cyclic nucleotides, hydrolysis of
phosphatidylinositol-~,5-biphosphate as well as the
activation of protein kinase C and an increase in the
intracellular calcium level. There is a great interest
in how these processes effect the metabolism of the cell
but at present they are hardly understood. A further
result of the binding of a ligand to its receptor can be
the migration of the receptor-ligand complex into the
inside of the cell by a receptor-dependent endocytosis.
This type of internalization apparently also occurs with
lymphokines (e.g. G-CSF), however, the consequences for
the metabolism of the cell are not yet understood.
Since G-CSF is able to substantially increase the
population of neutrophilic granulocytes within a short
period, considerable therapeutic fields of application
arise for G-CSF. Thus, G-CSF could be used e.g. after
chemotherapy in cancer, in which the cells of the immune
system are destroyed. In addition G-CSF could be used in
bone marrow transplantations, in severe burn wounds, in
opportunistic infections caused by immune deficiency and
in leukemia. For the different types of therapy it would
be desirable to develop more active and also less active
forrns of G-CSF. The object of the present invention is
therefore to develop G-CSF molecules with a wide
-`-` 20~1~39
-- 3 --
spectrum of activity by the specific introduction of
point mutations. In this~process the changes in activity
should be achieved by chanqes in the Dino acid sequence
which are as small as possible.
The object according to the present invention is
achieved by a granulocyte stimulating factor (G-CSF) or
a G-CSF variant, in which one or~several~amino acids of
the sequence Leu-Gly-His-Ser-Leu-Gly-Ile at position 50
to 56 of the mature G-CSF with~174 amino acids or at
position 53 to 59 of the mature G-CSF with 177 amino
acids or/and at least one of the 4 His residues at
position 43, 79, 156 or 170 of the mature G-CSF with 174
amino acids or at position 46, 82, 159 or 173 of the
mature G-CSF with 177 amino acids are mutagenized.
Surprisingly the introduction of new amino acids yields
G-CSF muteins which have a broad spectrum of activity.
The determination of the activity can for example be
carried out according to ~iochem. J. 253 (1988) 213-218;
Exp. Hematol. 17 (1989) }16-119; Proc. Natl. Acad. Sci.
USA 83 (1986) 5010.
The term G-CSF or G-CSF variant according to the present
invention includes all naturally occurring variants of
G-CSF with or without a leader sequence as well as G-CSF
proteins derived therefrom which are modified by
recombinant DNA technology, in particular fusion
proteins which contain further polypeptide sequences
apart from the G-CSF moiety. In this sense a G-CSF
mutein is particularly preferred with a N-terminal Met
residue at position -1 which is suitable for expression
in prokaryotic cells. Also preferred is a recombinant,
methionine-free G-CSF variant which can be produced
according to PCT/EP 91/00 192. The term "mutagenized"
~,
.
-`~` 2~4143g
- 4 -
means that the respective amino acid is deleted or
preferably substituted by another amino acid.
In this sense G-CSF muteins are preferred in which one
of the 7 amino acids of the sequence Leu-Gly-His-Ser-
Leu-Gly-Ile is substituted by another amino acid.
However, more than one, in particular two amino acids,
can also be replaced.
A G-CSF mutein is particularly preferred in which the
Ser residue at position 53 of the mature G-CSF with 174
amino acids or at position 56 of the mature G-CSF with
177 amino acids is replaced by one of the other 19 amino
acids, in particular by Thr.
Furthermore, it is preferred that the Leu residue at
position 54 of the mature G-CSF with 174 amino acids or
at position 57 of the mature G-CSF with 177 amino acids
is substituted by one of the 19 other amino acids, in
particular by Thr. By this means one obtains G-CSF
muteins with a broad variation of G-CSF activity.
In addition G-CSF muteins are preferred in which one of
the 4 His residues at position 43, 79, 156 or 170 of the
mature G-CSF with 174 amino acids or at position 46, 82,
159 or 173 of the mature G-CSF with 177 amino acids is
substituted by another amino acid, in particular Gln.
The invention also provides a recombinant DNA which
codes for a G-CSF mutein according to the present
invention. The invention also provides a recombinant
vector which contains at least one copy of a recombinant
DNA according to the present invention. In this
connection a recombinant vector is preferred which is
2041439
- 5 -
suitable for gene expression in prokaryotic cells.
vectors of this type are known to one skilled in the
art.
In addition the invention provides a celI which is
transformed with a recombinant DNA according to the
present invention or/and a recombinant vector according
to the present invention. This cell is preferably a
prokaryotic cell, particularly preferably an E. coli
cell.
The invention also provides a process for the production
of a recombinant DNA according to the present invention
in which a DNA sequence which codes for G-CSF or a G-CSF
variant is site-specifically mutagenized. The usual
molecular-biological methods for site-specific
mutagenesis are known to one skilled in the art. The
mutagenesis is preferably carried out by using synthetic
oligonucleotides as mutagenesis primers on singIe-
stranded DNA as the template. Common methods are for
example described in Amersham No. 1523 "Oligonucleotide-
; directed in vitro mutagenesis system"; Methods in
Enzymology (Academic Press, Inc. Vol. 154, Part E, 367-
382 (1987); Analytical Biochemistry 179 (1989) 309-311.
In addition the invention provides a process for
producing a G-CSF mutein according to the present
invention in which a cell is transformed with a
recombinant DNA according to the present invention
or/and a recombinant vector according to the present
invention, the transformed cell is cultured in a
suitable medium and the protein is isolated from the
cells or the medium. The methods usually used in
molecular biology for the isolation of recombinant
proteins from eukaryotic or prokaryotic cells are known
to one skilled in the art and do not need to be
elucidated in detail.
Finally the invention also provides a pharmaceutical
preparation based on a G-CSF mutein according to the
present invention as the active substance, if desired,
together with the usual pharmaceutical carrier, filling
and auxiliary substances. Such a pharmaceutical
preparation is particularly suitable for the therapeutic
fields of application mentioned above and ~ven for
further therapeutic proceedures in which the formation
of neutrophilic granulocytes is to be stimulated.
The following examples are intended to elucidate the
invention without however limiting its scope.
E x a m p l e
Production of the vector m~l-G-CSF-B~
The 554 bp long EcoRI/BamHI fragment from the vector pKK
177-3 G-CSF-Bg (DSM 5867) containing the Shine Dalgarno
sequence, ATG codon and coding sequence for the G-CSF
gene is cloned via a blunt-end ligation into the NcoI
cleavage site of the vector pPZ 07-mgl lac (W088/09373,
Figure lO). The ATG start codon of the lac Z gene, which
is located in the protruding single strand after NcoI
digestion, is digested beforehand by incubation with
mung bean nuclease (Pharmacia). The resulting vector is
denoted mgl-G-CSF-Bg.
-~` 2 0 ~ 3
-- 7
E x a m p 1 e 2
Mutagenesis of the amino acid Leu ~X) in the sequence
Gly-His-Ser-Leu-Gly-Ile
,~ :
The mutagenesis is carried out on the M13 template
according to known techniques (Amersham No. 1523
"Oligonucleotide-directed in vitro mutagenesis system").
A 251 bp long G-CSF cDNA fragment is isolated via the
cleavage site BstXI/AatII. The protruding single-strands
are digested off by mung bean nuclease (Pharmacia) and
the fragment is cloned into the vector M13mpl9 which was
cleaved with EcoRI/SmaI (EcoRI protruding single strand
was filled in for blunt-end cloning). After preparing
single-stranded DNA, the oligonucleotide is hybridized
to the single-stranded DNA and an elongation in the 5'-
3' direction beyond the oligonucleotide is carried out
using Klenow polymerase, ligase and the four nucleotide
triphosphates (GTP, CTP, TTP, ATP). The DNA which is now
double-stranded is transformed in E. coli cells which
carry a F' episome so that infection by filamentous M13
phages is possible (e.g. JM101, obtainable from
Stratagene, LaJolla, California). Individual plaques are
picked out and the mutagenized M13 phages contained
therein are used for the preparation of single-stranded
DNA. A DNA sequencing is carried out according to known
techniques (e.g. dideoxy method according to Sanger) and
the exact substitution to form the desired mutation is
checked in this way. After preparing double-stranded DNA
the mutated AvaI fragment of G-CSF is isolated and
cloned in the expression vector mgl-G-CSF-Bg (cleaved
with AvaI).
. .
2 ~
In order to reconstitute the complete G-CSF gene the DNA
is subsequently cleaved with HindIII, the protruding
ends are filled in with Klenow polymerase and afterwards
partially digested with AvaI so that the 5' AvaI site in
the G-CSF gene (at ca 130 bp) is not cleaved. This DNA
is ligated with the approximately 240 bp G-CSF fragment
AvaI/BamHI (BamHI site is filled in with Klenow
polymerase) from the starting vector mgl-G-CSF-Bg.
After transformation in E. coli JM83, the expression of
G-CSF is carried out in the manner described in
W~88/09373.
The cDNA used has a sequence which codes for a G-CSF
with 175 amino acids (without a signal sequence, but
with a Met residue at position -1) so that the preferred
mutation is located at Leu at position 54 of the G-CSF
amino acid sequence (in this the N-terminal Met residue
is not counted).
The sequence of the cDNA encoding G-CSF which codes for
the amino acids 50 to 56 (with reference to the G-CSF
with 174 amino acids) reads:
(X)
Leu-Gly-His-Ser-Leu-Gly-Ile
5'-CTC GGA CAC TCT CTG GGC ATC-3'
The corresponding complementary opposite strand to be
mutagenized reads:
5l-GAT GCC CAG AGA GTG TCC GAG-3'
20~1~}~3
g
The following 19 oligonucleotides correspond:ing to the
opposite strand are used for site-directed mutagenesis:
Wild-type: 5'-3 GAT GCC CAG AGA GTG TCC~GAG 3'
Met
l. S' GAT GCC CAT AGA GTG TCC GAG 3'
:
Phe
2. 5' GAT GCC GAA AGA GTG TCC GAG 3'
Gln
3. 5' GAT GCC CTG AGA GTG TCC GAG 3'
^: Glu
4. 5' GAT GCC CTC AGA GTG TCC GAG :3'
Asp
~ 5. 5' GAT GCC GTC AGA GTG TCC GAG 3'
,~ : Cys
6. 5' GAT GCC GCA AGA GTG TCC GAG 3'
, ~ ,
~; Ala
7. 5' GAT GCC GGC AGA GTG TCC GAG 3'
~ .~
Gly
8. 5' GAT GCC AGG AGA GTG TCC GAG 3'
His
9. 5' GAT GCC GTG AGA GTG TCC GAG 3'
Ile
10. 5' GAT GCC GAT AGA GTG TCC GAG 3'
~,
-- 10 --
.
Lys
11. 5' GAT GCC CTT AGA GTG TCC GAG 3'
Tyr
12. 5I GAT GCC ATA AGA GTG TCC GAG 3'
Asn
13. 5' GAT GCC GTT AGA GTG TCC GAG 3'
Pro
14. 5' GAT GCC GGG AGA GTG TCC GAG 3'
Arg
15. 5' GAT GCC GCG AGA GTG TCC GAG 3'
Ser
16. 5' GAT GCC GGA AGA GTG TCC GAG 3'
Thr
17. 5' GAT GCC GGT AGA GTG TCC GAG 3'
Val
18. 5' GAT GCC GAC AGA GTG TCC GAG 3'
Trp
19. 5' GAT GCC CCA AGA GTG TCC GAG 3'
E x a m p l e 3
Productlon_of a G-CSF with modified activity
A G-CSF which is more enzymatically active compared to
the wild type can be produced by substituting serine at
position 53 by a threonine at position 53 of a G-CSF
with 174 amino acids (serine in the sequence Gly-His-
-` 20~14~9
11 --
Ser-Leu-Gly). The following double-stranded
oligonucleotide was used for the mutagenesis:
His Thr Leu Gly Ile
S' CCC GAG GAG CTG GTG CTG CTC GGA CAC ACC CTG GGC ATC CCC TGG GCT CCC CTG AGC 3'
3' C CTC GAC CAC GAC GAG CCT GTG TGG GAC CCG TAG GGG ACC CGA GGG GAC 5'
For the cloning, the G-CSF cDNA fragment (ca 300 bp,
EcoRI/EcoRV) from the vector pKK 177-3 G-CSF-Bg (DSM
5867) was ligated into the EcoRI/SmaI cleavage site of
the vector pUC19 (Yannish-Perron et al., (1985), Gene
33, 103).
This DNA is cleaved with AvaI/SacI and directly ligated
with the primer pair described above according to the
usual techniques. The mutated BstIXlSacI fragment can
now be isolated from this construct and cloned into the
vector pXK 177-3 G-CSF-Bg (DSM 5867) (cleaved with
BstXI/SacI). The final construction of the expression
clone is carried out in analogy to Example l. The
determination of activity is carried out as described in
Example 5.
E x a m p l e 4
Alteration of ,t~,he enzvmatic properties of G-CSF_by
mutation of amin,o acids which are not located in the
active_centre.
In analogy to known serine esterases it is assumed that
the serine of the active centre interacts with histidine
for the development of enzymatic activity. Four
histidines are present in the sequence of G-CSF and
namely at positions 43, 79, 156 and 170 (numbered from
~-` 2 0 ~ 9
- 12 -
the 174 aa sequence without a signal peptide~. The
histidine residue at position 52 (or at position 55 in
the 177 amino acid form) is left out of consideration in
this mutagenesis. In this process His (CCA, CTA) is
substituted by Gln (CAG). The sequence on the opposite
strand corresponding to the codon coding for Gln is CTG.
:`
A G-CSF fragment is subcloned in M13mpl9 as described in
Example 1.
The following~oligonucleotides corresponding to the
opposite strand are used for the mutagenesis:
l. 5' GCT CCT GGG CTG GCA CAG C 3'
histidine 43 to glutamine 43
2. 5' GAA AAG GCC GCT CTG GAG TTG GCT C 3'
histidine 79 to glutamine 79
;: : :
3. 5' GCT CTG CAG CTG GCC TAG CAA CC 3'
histidine 156 to glutamine 156
4. 5' GGG CTG CGC AAG CTG GCG TAG AAC G 3
histidine 170 to glutamine 170
f,
~ ;
The analytical procedure and the recloning in an
expression vector is carried out in analogy to Example
1.
:
~ 20~3~
- 13 -
E x a m p 1 e 5
Determination of the G-~SF act;ivity
The ac~tivity of G-CSF is tested with the murine
leukaemia line NSF60 which is completely dependent on G-
CSF as described in Biochem. J. 253 (1988) ~213-218, Exp.
Hematol. 17 (1989) 116-119, Proc. Natl. Acad. Sci. USA
83 (1986) 5010. In order that the factor-dependency of
the cells is preserved, the medium (RPMI medium,
Boehringer Mannheim GmbH, Order No. 2099445 with 10 %
foetal calf serum) for the maintenance culture
permanently contains 1000 U/ml G-CSF.
The proliferation of the NSF60 cells stimulated by G-CSF
is measured directly in this test by the incorporation
of 3H thymidine. The test is carried out as follows:
NSF60 cells which are in the exponential growth phase
(cell density is maximally lx105 cells/ml) are
transferred to microtitre plates (lx104 celIs/well) and
cuItured with a decreasing G-CSF concentration. The
maximum dose of G-CSF in well 1 corresponds to the
concentration in the maintenance culture (1000 U/ml,
specific activity lx108 U/mg protein). The dilution is
carried out in steps of ten.
After about 24 hours incubation 3H thymidine
(0.1 ~Ci/well) is added. Afterwards the cells are
incubated for a further 16 hours.
In order to evaluate the test the cells in the
microtitre plates are frozen in order to lyse them. The
cell lysate is aspirated on a glass fibre filter,
20~3~
-- 14 --
rinsed, dried and measured in a scintillation counter.
The incorporation of 3H thymidine is proportional to the
G-CSF-induced proliferation of the NSF60 cells.
E x a m p 1 e 6
,
Alt ration i~__he activity of G-CSF by_amino acid
substitution in the active centre
A G-CSF modified in amino acid position 54 can be
produced by substitution of preferably one leucine at
position 54 by a threonine at position 54 (Leu in the
sequence Gly-His-Ser-Leu-Gly) in correspondence with the
procedure described in Example 3 using a suitable
double-stranded oligonucleotide which contains a nucleic
acid triplet (e.g. ACC) coding for the amino acid Thr at
the appropriate position. In this connection position 54
of the 174 amino acid form of G-CSF corresponds to
position 57 of the 177 amino acid form.
The activity of a mutant having 174 amino acids with Thr
at position 54 is reduced in the NSF60 cell test (see
Example 5) in comparison to the wild-type G-CSF with 174
amino acids. Moreover, the activity of this G-CSF mutant
is reduced in comparison to a G-CSF mutant with an amino
acid substitution of a serine by a threonine at position
53 (described in Example 3).
