m Introduction:

. What are CSFǯS?

. What is G-CSF?
. What is HG-CSF?
. Biological function of G-CSF
. Therapeutic uses of G-CSF

d Binding and action of G-CSF.

m Structure of KW-2228.
m Comparisons and differences between wild type. G-
CSF and KW-2228.
m Stability and activity of KW-2228.
m Conclusion .
m Colony-stimulating factors (CSFs) are a family of
glycoproteins that stimulate the proliferation and
differentiation of hematopoietic cells in vivo.

TYPES OF CSFǯS:
m Granulocyte colony-stimulating factor (G-CSF),
m Granulocyte-macrophage colony-stimulating
factor (GM-CSF),
m Macrophage colony-stimulating factor (M-CSF),
m Interleukins and Erythropoietin.
m Granulocyte colony-stimulating factor (G-CSF or
GCSF) is a colony-stimulating factor hormone.
m It is a 19.6-kDa glycoprotein consisting of 174 amino
acid residues, produced by a number of different
tissues to stimulate the bone marrow to produce
granulocytes and stem cells.
m G-CSF then stimulates the bone marrow to release
them into the blood.
m It also stimulates the survival, proliferation,
differentiation, and function of neutrophil precursors
and mature neutrophils.
m G-CSF is produced by endothelium, macrophages, and a
number of other immune cells.
m The natural human glycoprotein exists in two forms, a 174-
and 180-amino-acid-long protein of molecular weight
19,600 grams per mole.
m The more-abundant and more-active 174-amino acid form
has been used in the development of pharmaceutical
products by recombinant DNA (rDNA) technology.
m The G-CSF-receptor is present on precursor cells in the
bone marrow, and, in response to stimulation by G-CSF,
initiates proliferation and differentiation into mature
granulocytes.
m G-CSF is a also a potent inducer of HSCs(Hematopoeitic
Stem Cells) mobilization from the bone marrow into the
bloodstream.
m Human G-CSF (hG-CSF) is a 19.6-kDa glycoprotein
consisting of 174 amino acid residues
m Human granulocyte colony stimulating factor
(hG-CSF) is a hematopoietic cytokine that acts on
cells of the neutrophil lineage causing
proliferation and differentiation of committed
precursor cells and activation of mature
neutrophils.
m KW-2228 is a tailored human granulocyte colony-
stimulating factor (hG-CSF) which has more
potent granulopoietic activity and is more stable
than wild-type hG-CSF.
m More than 100 novel hG-CSFs have been
engineered with the aim of identifying novel
proteins with enhanced biological activity and
physicochemical properties .
m One of these engineered proteins, designated
{
, has more potent granulopoietic activity
than that of wild-type hG-CSF, both in vitro and in
vivo .
m KW-2228 (Neu-up) is currently being used
clinically and is yielding very satisfactory results.
m G-CSF stimulates the production of white blood cells (WBC).
m In oncology and hematology, a recombinant form of G-CSF is used with
certain cancer patients to accelerate recovery from neutropenia after
chemotherapy, allowing higher-intensity treatment regimens.

m Chemotherapy can cause myelosuppression and unacceptably low levels

of white blood cells, making patients prone to infections and sepsis.

m G-CSF is also used to increase the number of hematopoietic stem

m cells in the blood of the donor before collection by leukapheresis for use
in hematopoietic stem cell transplantation.
m It may also be given to the receiver, to compensate for conditioning
regimens.
m G-CSF is used to treat heart degeneration by injecting it into the blood-
stream, plus SDF(stromal cell-derived factor) directly to the heart.

m Recently, the application of G-CSF against infectious diseases has been

considered, and some animal experiments have been carried out to
support such an application on human infectious diseases.
m Sweet's syndrome is a known side effect of using
this drug.
m It was first marketed by Amgen with the brand
name Neupogen.
m The recombinant human G-CSF synthesised in an
E. coli expression system is called filgrastim.
m Filgrastim (Neupogen) and PEG-filgrastim
(Neulasta) are two commercially-available forms
of rhG-CSF (recombinant human G-CSF).
m Another form of recombinant human G-CSF
called lenograstim is synthesised in Chinese
Hamster Ovary cells (CHO cells).
m The binding of G-CSF to its receptor on pluripotent stem
cells triggers terminal differentiation of the cells.
m Understanding the binding mechanism is a prerequisite to
designing an improved G-CSF.
m The residues that contribute to G-CSF receptor binding are
located on the surface of G-CSF.
m They predicted that residues 20Ȃ46 and the C-terminus
bind to the G-CSF receptor.
m Residues 138Ȃ142 are recognized by common antibodies.
m Based on the structural and biological information
obtained to date, we predict that residues 43Ȃ46 and 138Ȃ140
and/or those near them are involved in G-CSF receptor
binding .
m The main structural feature of KW-2228 is an antiparallel four-Ʉ-
helix bundle (Fig. 1 and Fig. 2) which has a left-handed twist with
overall dimensions of approximately 45×25×25 Å3.

m The Ʉ-helices, which are designated A through D, consist of

residues 10Ȃ39, 73Ȃ92, 99Ȃ123, and 142Ȃ171, respectively.

m The overall topology of the bundle is very similar to those

observed in bovine, canine and wild-type hG-CSFs and very close
to that of an ideal left-handed antiparallel four-Ʉ-helix bundle.

m The four helices are connected by three loops designated AB, BC

and CD. The AB and CD loops are long overhand connections,
and the shortest loop, BC, joins helix B to helix C. In addition to
the major helices there is a short extended 310 helix designated E
within the AB loop which consists of residues 43Ȃ52.
à
 
   
   

 

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6G-CSF (which is the G-CSF structure most
accurately determined so far), the coordinates of
residues 1Ȃ7, 129Ȃ135 and 174 are not located clearly
and the structures of the N-terminal region and
the middle of the CD loop are not definitive.
m In the structure of KW-2228, however, the electron
density is clearly defined and the positions of all
of the residues except 1, 2, 130 and 131 are
definitive.
m Thr1, Leu3, Gly4, Pro5 and Cys17 in wild-type hG-CSF
are replaced by Ala, Thr, Tyr, Arg and Ser,
respectively, in KW-2228.
m Although G-CSF is expected to be structurally
flexible due to the presence of the remarkably
long overhand loops, the substitution of amino
acids at residues 1, 3, 4, 5 and 17 in KW-2228 has
frozen the conformation and allowed us to
determine almost the entire structure of hG-CSF
for the first time.
m There are two disulfide bridges in hG-CSF. The
Cys64ȂCys74 disulfide bridge is buried in the
interior of the molecule, but the Cys36ȂCys42
bridge is on the surface of the molecule. These
disulfide bridges restrict the conformation of the
long overhand AB connection.

m There is a specific hydrophobic network in the

interior of the bundle which is markedly rich in
leucine residues.
m The characteristic leucine-rich hydrophobic
network obviously plays a very important role in
stabilization of the four helices in the bundle
configuration.

m In G-CSF there are particularly long overhand

connections which are potentially flexible.
Therefore the leucine-rich hydrophobic network
may function as a scaffold to stabilize the bundle
and adjust the positions of specific residues
favorable for binding of receptors.
m The biological activity and stability of KW-2228 in
human serum are markedly enhanced compared with
those of wild-type hG-CSF.
m The residues which are engineered in KW-2228 are
located near the N-terminus.
m The positively charged atom of Arg5 is located near the
carbonyl oxygen of Ala172
m The specific network of interactions which involve
Arg5 contribute stabilization of the conformation of
the N- and C-terminal regions and fix the middle of
the long overhand AB connection in KW 2228.
m Cys17 is substituted by Ser in KW-2228.
m The hydroxyl group of Ser17 forms a hydrogen bond
with the carbonyl group of Leu14 in kw-2228.
m This substitution may contribute to the stability of the
protein.
In short,
m The mutations introduced in KW-2228 contribute to
the stabilization of the conformation of the molecule
including the two long overhand connections.
m The significant enhancement of the physicochemical
and biological stability and biological activity of KW-
2228 is undoubtedly due to its stabilized active
conformation.
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m 2 nk nghub.e2sev er.com/retr eve/p /S0014579397005383 Ȃ
m www.ncb .n2m.n h.gov/
m www.youtube.com/watch
m Isao Fuj
Yosh tomo Nagahara
Motoo Yamasak
Yosh haru Yokoo
Se ga Itoh
and Nor ak  rayama .Department of B o2og ca2 Sc ence and Techno2ogy
Toka
Un vers ty
317 N sh no
Numazu
Sh zuoka 410-03
Japan
Tokyo Research Laborator es
Kyowa akko Kogyo Co. Ltd.
3-6-6 Asah mach

Mach da
Tokyo 194
Japan.
m www.sc enced rect.com/sc ence?_ob=Art c2eURL&_ud =B6T36-3RB8YT6-
2B&_user=10&_rdoc=1&_fmt=&_or g=search&_sort=d&_docanchor=&v ew=c&
_searchStrId=1002387513&_rerunOr g n=goog2e&_acct=C000050221&_vers on=1
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