The invention relates to granulocyte colony stimulating factor (GCSF) fusion polypeptides
and dimers; nucleic acid molecules encoding said polypeptides and methods of treatment
that use said proteins/dimers.
Cytokine receptors can be divided into three separate groups. Class 1 (referred to as
the haemotopoietin or growth hormone family) receptors are characterised by four
conserved cysteine residues in the amino terminal part of their extracellular domain and
the presence of a conserved Trp-Ser-Xaa-Trp-Ser motif in the C-terminal part. The
receptors consist of two polypeptide chains. Class I receptors can be sub-divided into the
GM-CSF sub-family (which includes IL-3, IL-5, GM-CSF, GCSF) and IL-6 sub-family
(which includes IL-6, IL-11 and IL-12). In the IL-6 sub-family there is a common
tranduscing subunit (gp130) that associates with one or two different cytokine subunits.
There is a further sub-family referred to as the IL-2 sub-family (includes IL-2, IL-4, IL-7,
IL-9 and IL-15. The repeated Cys motif is also present in Class 2 (interferon receptor
family) the ligands of which are α, β and γ interferons but lack the conserved Trp-Ser-
Xaa-Trp-Ser motif.
GCSF stimulates the proliferation and differentiation of granulocyte progenitor cells.
GCSF is encoded by a single gene that encodes two polypeptides that result from
differential splicing of mRNA. The polypeptides are 177 and 180 amino acids in length
with the mature polypeptide having a molecular weight of 19.6kD. GCSF is produced by
the endothelium and macrophages and acts through the GCSF receptor (GCSFR) which
is expressed on granulocyte progenitor cells in bone marrow which when activated
results in their maturation into granulocytes. These can then differentiate into neutrophil
precursors and mature neutrophils. The main therapeutic application of recombinant
GSCF is in the treatment of patients undergoing chemotherapy for cancer which results
in the loss of neutrophils and consequently the development of neutropenia. Neutropenia
results in immune suppression and exposure of the patient to infection and sepsis. In
addition recombinant GCSF is used to increase the number of haematopoietic stem cells
in vivo prior to harvesting and use in haematopoietic stem cell transplantation.
This disclosure relates to the identification of GCSF recombinant forms that have
improved pharmacokinetics (PK) and activity. The new GCSF molecules have biological
activity, form dimers and have improved stability.

According to an aspect of the invention there is provided a nucleic acid molecule
comprising a nucleic acid sequence that encodes a polypeptide having the activity of
granulocyte colony stimulating factor comprising a granulocyte colony stimulating factor
polypeptide linked, directly or indirectly, to at least one cytokine binding domain of the
granulocyte colony stimulating factor receptor polypeptide.
According to an aspect of the invention there is provided a fusion polypeptide comprising:
the amino acid sequence of granulocyte colony stimulating factor polypeptide, or active
part thereof linked, directly or indirectly, to at least one cytokine binding domain of the
granulocyte colony stimulating factor receptor polypeptide.
In a preferred embodiment of the invention said fusion polypeptide comprises two
cytokine homology binding domains of the granulocyte colony stimulating factor receptor
polypeptide.
In a further preferred embodiment of the invention said fusion polypeptide further
comprises an immunoglobulin-like domain.
In a further preferred embodiment of the invention said fusion polypeptide includes at
least one fibronectin domain III; preferably two or three fibronectin III domains.
The GCSFR is complex comprises a series of domains that contribute to its molecular
structure. GCSFR can be sub-divided into several regions that are structurally and
functionally defined. The receptor is 812 amino acids in length and is typical of cytokine
receptors in so far as it includes an extracellular domain, a single transmembrane domain
and a cytoplasmic domain. The extracellular domain has a modular structure comprising
from the amino terminus in the mature polypeptide; an immunoglobulin-like domain
(amino acids 1-97); a first cytokine homology domain (97-201) and second cytokine
domain (202-313) and three fibronectin III domains. Functionally the first and second
cytokine domains bind GCSF.
In a preferred embodiment of the invention said fusion polypeptide comprises amino acid
residues 97-201 as represented in SEQ ID NO: 31

'In an alternative preferred embodiment of the invention said fusion polypeptide comprises
amino acid residues 202-313 of SEQ ID NO: 31.
In an alternative preferred embodiment of the invention said fusion polypeptide comprises
amino acid residues 97-313 of SEQ ID NO: 31.
In a further preferred embodiment of the invention said fusion polypeptide comprises
amino acid residues 1-97 of SEQ ID NO: 31.
In a preferred embodiment of the invention polypeptide is linked to the cytokine binding
domain wherein said granulocyte colony stimulating factor polypeptide is positioned
amino terminal to said cytokine binding domain in said fusion polypeptide.
In an alternative preferred embodiment of the invention granulocyte colony stimulating
factor polypeptide is linked to the cytokine binding domain wherein said granulocyte
colony stimulating factor polypeptide is positioned carboxyl-terminal to said cytokine
binding domain in said fusion polypeptide.
In a preferred embodiment of the invention granulocyte colony stimulating factor is linked
to the binding domain of the granulocyte colony stimulating factor receptor polypeptide by
a peptide linker; preferably a flexible peptide linker.
In a preferred embodiment of the invention said peptide linking molecule comprises at
least one copy of the peptide Gly Gly Gly Gly Ser.
In a preferred embodiment of the invention said peptide linking molecule comprises 2, 3,
4, 5, 6, 7, 8, 9 or 10 copies of the peptide Gly Gly Gly Gly Ser.
Preferably said peptide linking molecule consists of 6 copies of the peptide Gly Gly Gly
Gly Ser.
In an alternative embodiment of the invention said polypeptide does not comprise a
peptide linking molecule and is a direct fusion of granulocyte colony stimulating factor
polypeptide and the binding domain of granulocyte colony stimulating factor receptor
polypeptide.

According to an aspect of the invention there is provided a nucleic acid molecule
comprising a nucleic acid sequence selected from:
i) a nucleic acid sequence as represented in SEQ ID NO:5;
ii) a nucleic acid sequence as represented in SEQ ID NO 7:;
iii) a nucleic acid sequence as represented in SEQ ID NO: 9;
iv) a nucleic acid sequence as represented in SEQ ID NO:11;
v) a nucleic acid sequence as represented in SEQ ID NO: 13;
vi) a nucleic acid sequence as represented in SEQ ID NO: 15;
vii) a nucleic acid sequence as represented in SEQ ID NO: 17;
viii) a nucleic acid sequence as represented in SEQ ID NO: 19; or
a nucleic acid molecule comprising a nucleic sequence that hybridizes under stringent
hybridization conditions to SEQ ID NO:5-SEQ ID NO: 19 and which encodes a
polypeptide that has granulocyte colony stimulating factor receptor modulating activity.
In a preferred embodiment of the invention said nucleic acid molecule encodes a
polypeptide that has agonist activity.
In a preferred embodiment of the invention said nucleic acid molecule encodes a
polypeptide that has antagonist activity.
Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid
molecules undergo an amount of hydrogen bonding to each other. The stringency of
hybridization can vary according to the environmental conditions surrounding the nucleic
acids, the nature of the hybridization method, and the composition and length of the
nucleic acid molecules used. Calculations regarding hybridization conditions required for
attaining particular degrees of stringency are discussed in Sambrook et al., Molecular
Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology—Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York,
1993). The Tm is the temperature at which 50% of a given strand of a nucleic acid
molecule is hybridized to its complementary strand. The following is an exemplary set of
hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize)
Hybridization: 5x SSC at 65°C for 16 hours
Wash twice: 2x SSC at room temperature (RT) for 15 minutes each
Wash twice: 0.5x SSC at 65°C for 20 minutes each
High Stringency (allows seguences that share at least 80% identity to hybridize)
Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours
Wash twice: 2x SSC at RT for 5-20 minutes each
Wash twice: 1 x SSC at 55°C-70°C for 30 minutes each
Low Stringency (allows seguences that share at least 50% identity to hybridize)
Hybridization: 6x SSC at RT to 55°C for 16-20 hours
Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.
In a preferred embodiment of the invention said nucleic acid molecule comprises or
consists of a nucleic acid sequence as represented in SEQ ID NO: 5.
In a preferred embodiment of the invention said nucleic acid molecule comprises or
consists of a nucleic acid sequence as represented in SEQ ID NO: 7.
In a preferred embodiment of the invention said nucleic acid molecule comprises or
consists of a nucleic acid sequence as represented in SEQ ID NO: 9.
In a preferred embodiment of the invention said nucleic acid molecule comprises or
consists of a nucleic acid sequence as represented in SEQ ID NO: 11.
In a preferred embodiment of the invention said nucleic acid molecule comprises or
consists of a nucleic acid sequence as represented in SEQ ID NO: 13.
In a preferred embodiment of the invention said nucleic acid molecule comprises or
consists of a nucleic acid sequence as represented in SEQ ID NO: 15.
In a preferred embodiment of the invention said nucleic acid molecule comprises or
consists of a nucleic acid sequence as represented in SEQ ID NO: 17.

'in a preferred embodiment of the invention said nucleic acid molecule comprises or
consists of a nucleic acid sequence as represented in SEQ ID NO: 19.
According to an aspect of the invention there is provided a polypeptide encoded by the
nucleic acid according to the invention.
According to a further aspect of the invention there is provided a polypeptide comprising
or consisting of an amino acid sequence selected from the group consisting of: SEQ ID
NO: 6, 8, 10, 12, 14, 16, 18, 20, 25, 26, 27, 28, 29 or 30.
In a preferred embodiment of the invention said polypeptide has agonist activity.
In an alternative preferred embodiment of the invention said polypeptide has antagonist
activity.
According to an aspect of the invention there is provided a homodimer consisting of two
polypeptides wherein each of said polypeptides comprises:
i) a first part comprising granulocyte colony stimulating factor, or a receptor
binding domain thereof, optionally linked by a peptide linking molecule to
ii) a second part comprising the cytokine homology binding domain or part
thereof, of the granulocyte colony stimulating factor receptor.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 6.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 8.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 10.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 12.

'In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 14.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 16.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 18.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 20.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 25.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 26.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 27.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 28.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 29.
In a preferred embodiment of the invention said homodimer comprises two polypeptides
comprising or consisting of SEQ ID NO: 30.
According to a further aspect of the invention there is provided a vector comprising a
nucleic acid molecule according to the invention.
In a preferred embodiment of the invention said vector is an expression vector adapted to
express the nucleic acid molecule according to the invention.

A vector including nucleic acid (s) according to the invention need not include a promoter
or other regulatory sequence, particularly if the vector is to be used to introduce the
nucleic acid into cells for recombination into the genome for stable transfection.
Preferably the nucleic acid in the vector is operably linked to an appropriate promoter or
other regulatory elements for transcription in a host cell. The vector may be a bi-
functional expression vector which functions in multiple hosts. By "promoter" is meant a
nucleotide sequence upstream from the transcriptional initiation site and which contains
all the regulatory regions required for transcription. Suitable promoters include
constitutive, tissue-specific, inducible, developmental or other promoters for expression in
eukaryotic or prokaryotic cells. "Operably linked" means joined as part of the same
nucleic acid molecule, suitably positioned and oriented for transcription to be initiated
from the promoter. DNA operably linked to a promoter is "under transcriptional initiation
regulation" of the promoter.
In a preferred embodiment the promoter is a constitutive, an inducible or regulatable
promoter.
According to a further aspect of the invention there is provided a cell transfected or
transformed with a nucleic acid molecule or vector according to the invention.
Preferably said cell is a eukaryotic cell. Alternatively said cell is a prokaryotic cell.
In a preferred embodiment of the invention said cell is selected from the group consisting
of; a fungal cell (e.g. Pichia spp, Saccharomyces spp, Neurospora spp); insect cell (e.g.
Spodoptera spp); a mammalian cell (e.g. COS cell, CHO cell); a plant cell.
In a preferred embodiment of the invention said cell is stably transfected or transformed.
According to a further aspect of the invention there is provided a pharmaceutical
composition comprising a polypeptide according to the invention including an excipient or
carrier.
In a preferred embodiment of the invention said pharmaceutical composition is combined
with a further therapeutic agent.

When administered the pharmaceutical composition of the present invention is
administered in pharmaceutically acceptable preparations. Such preparations may
routinely contain pharmaceutically acceptable concentrations of salt, buffering agents,
preservatives, compatible carriers, and optionally other therapeutic agents.
The pharmaceutical compositions of the invention can be administered by any
conventional route, including injection. The administration and application may, for
example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, intra-articuar,
subcutaneous, topical (eyes), dermal (e.g a cream lipid soluble insert into skin or mucus
membrane), transdermal, or intranasal.
Pharmaceutical compositions of the invention are administered in effective amounts. An
"effective amount" is that amount of pharmaceuticals/compositions that alone, or together
with further doses or synergistic drugs, produces the desired response. This may involve
only slowing the progression of the disease temporarily, although more preferably, it
involves halting the progression of the disease permanently. This can be monitored by
routine methods or can be monitored according to diagnostic methods.
The doses of the pharmaceuticals compositions administered to a subject can be chosen
in accordance with different parameters, in particular in accordance with the mode of
administration used and the state of the subject (i.e. age, sex). When administered, the
pharmaceutical compositions of the invention are applied in pharmaceutically-acceptable
amounts and in pharmaceutically-acceptable compositions. When used in medicine salts
should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may
conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically and pharmaceutically-
acceptable salts include, but are not limited to, those prepared from the following acids:
hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric,
formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be
prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium
salts.
The pharmaceutical compositions may be combined, if desired, with a pharmaceutically-
acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein

means one or more compatible solid or liquid fillers, diluents or encapsulating substances
that are suitable for administration into a human. The term "carrier" denotes an organic
or inorganic ingredient, natural or synthetic, with which the active ingredient is combined
to facilitate the application. The components of the pharmaceutical compositions also are
capable of being co-mingled with the molecules of the present invention, and with each
other, in a manner such that there is no interaction that would substantially impair the
desired pharmaceutical efficacy.
The pharmaceutical compositions may contain suitable buffering agents, including: acetic
acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
The pharmaceutical compositions also may contain, optionally, suitable preservatives,
such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
The pharmaceutical compositions may conveniently be presented in unit dosage form
and may be prepared by any of the methods well-known in the art of pharmacy. All
methods include the step of bringing the active agent into association with a carrier that
constitutes one or more accessory ingredients. In general, the compositions are
prepared by uniformly and intimately bringing the active compound into association with a
liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the
product.
Compositions suitable for oral administration may be presented as discrete units, such as
capsules, tablets, lozenges, each containing a predetermined amount of the active
compound. Other compositions include suspensions in aqueous liquids or non-aqueous
liquids such as syrup, elixir or an emulsion.
Compositions suitable for parenteral administration conveniently comprise a sterile
aqueous or non-aqueous preparation that is preferably isotonic with the blood of the
recipient. This preparation may be formulated according to known methods using
suitable dispersing or wetting agents and suspending agents. The sterile injectable
preparation also may be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butane diol.
Among the acceptable solvents that may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally

employed as a solvent or suspending medium. For this purpose any bland fixed oil may
be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as
oleic acid may be used in the preparation of injectables. Carrier formulation suitable for
oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
According to a further aspect of the invention there is provided a method to treat a
human subject suffering from a condition that would benefit from administration of a
granulocyte colony stimulating factor agonist comprising administering an effective
amount of at least one polypeptide according to the invention.
In a preferred method of the invention said polypeptide is administered intravenously.
In an alternative preferred method of the invention said polypeptide is administered
subcutaneously.
In a further preferred method of the invention said polypeptide is administered at two day
intervals; preferably said polypeptide is administered at weekly, 2 weekly or monthly
intervals.
In a preferred method of the invention said condition is neutropenia.
According to a further aspect of the invention there is provided a method to stimulate
haematopoietic progenitor cell proliferation and/or differentiation in a human subject
comprising administering an effective amount of at least one polypeptide according to the
invention.
In a preferred method of the invention said method is an in vitro method.
In an alternative preferred method of the invention said method is an in vivo method.
In a preferred method of the invention following stimulation of haematopoietic progenitor
cells bone marrow is harvested from said human subject and used for haematopoietic
progenitor cell transplantation.

Preferably said harvested bone marrow is administered to a human subject in need of
bone marrow transplantation.
According to an aspect of the invention there is provided the use of a polypeptide
according to the invention for the manufacture of a medicament for the treatment of
neutropenia.
In a further preferred embodiment of the invention said polypeptide is administered at two
day intervals; preferably said polypeptide is administered at weekly, 2 weekly or monthly
intervals.
According to a further aspect of the invention there is provided the use of an effective
amount of a polypeptide according to the invention in the manufacture of a medicament
for the stimulation of haematopoietic progenitor cell of proliferation and/or differentiation
in a human subject.
According to a further aspect of the invention there is provided a monoclonal antibody
that binds the polypeptide or dimer according to the invention.
Preferably said monoclonal antibody is an antibody that binds the polypeptide or dimer
but does not specifically bind granulocyte colony stimulating factor or granulocyte colony
stimulating factor receptor individually.
The monoclonal antibody binds a conformational antigen presented either by the
polypeptide of the invention or a dimer comprising the polypeptide of the invention.
In a further aspect of the invention there is provided a method for preparing a hybridoma
cell-line producing monoclonal antibodies according to the invention comprising the steps
of:
i) immunising an immunocompetent mammal with an immunogen
comprising at least one polypeptide according to the invention;
ii) fusing lymphocytes of the immunised immunocompetent mammal with
myeloma cells to form hybridoma cells;
iii) screening monoclonal antibodies produced by the hybridoma cells of step
(ii) for binding activity to the polypeptide of (i);

iv) culturing the hybridoma cells to proliferate and/or to secrete said
monoclonal antibody; and
v) recovering the monoclonal antibody from the culture supernatant.
Preferably, the said immunocompetent mammal is a mouse. Alternatively, said
immunocompetent mammal is a rat.
The production of monoclonal antibodies using hybridoma cells is well-known in the art.
The methods used to produce monoclonal antibodies are disclosed by Kohler and
Milstein in Nature 256, 495-497 (1975) and also by Donillard and Hoffman, "Basic Facts
about Hybridomas" in Compendium of Immunology V.ll ed. by Schwartz, 1981, which are
incorporated by reference.
According to a further aspect of the invention there is provided a hybridoma cell-line
obtained or obtainable by the method according to the invention.
According to a further aspect of the invention there is provided a diagnostic test to detect
a polypeptide according to the invention in a biological sample comprising:
i) providing an isolated sample to be tested;
ii) contacting said sample with a ligand that binds the polypeptide or dimer
according to the invention; and
iii) detecting the binding of said ligand in said sample.
In a preferred embodiment of the invention said ligand is an antibody; preferably a
monoclonal antibody.
Throughout the description and claims of this specification, the words "comprise" and
"contain" and variations of the words, for example "comprising" and "comprises", means
"including but not limited to", and is not intended to (and does not) exclude other
moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the
plural unless the context otherwise requires. In particular, where the indefinite article is
used, the specification is to be understood as contemplating plurality as well as
singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in
conjunction with a particular aspect, embodiment or example of the invention are to be
understood to be applicable to any other aspect, embodiment or example described
herein unless incompatible therewith.
An embodiment of the invention will now be described by example only and with
reference to the following figures:
Figure 1a nucleic acid sequence encoding GCSF expressed in a mammalian cell line.
Signal sequence is shown in bold and lower case, "refers to stop codon. Nucleotide
length = 522bp, (not including signal sequence); Figure 1b amino acid sequence length =
174aa (not including signal sequence);
Figure 2a nucleic acid sequence encoding GCSF expressed in E. coli (pET21a (+)), with
6x Histidine tag; ATG start codon in bold. *refers to stop codon. Letters in bold italics
refer to excess sequence at 5' end due to Hist-tag; Figure 2b mature amino acid
sequence length = 182aa (not including methionine start);
Figure 3a nucleic acid sequence encoding GCSF-L6-GCSFrEC (1-3): contains GCSF
linked via G4Sx6 to GCSF extracellular receptor domains 1-3 (Ig, BN and BC). *refers to
stop codon. Signal sequence in bold and lower case; Figure 3b amino acid sequence
length = 511aa (not including signal sequence);
Figure 4a nucleic acid sequence encoding GCSF-L6-GCSFrEC (1-3) expressed in E.
coli: contains GCSF linked via G4Sx6 to GCSF extracellular receptor domains 1-3 (Ig, BN
and BC). "refers to stop codon; ATG start codon in bold. *refers to stop codon. Letters in
bold italics refer to excess sequence at 5' end due to Hist-tag; Figure 4b amino acid
sequence length = 519aa (not including Methionine start);
Figure 5a nucleic acid sequence encoding GCSF-L8-GCSFrEC (1-3) expressed in a
mammalian cell line: contains GCSF linked via G4Sx8 to GCSF extracellular receptor
domains 1-3 (Ig, BN and BC). *refers to stop codon. Signal sequence in bold and lower
case; Figure 5b amino acid sequence length = 521 aa (not including signal sequence)

'Figure 6a nucleic acid sequence encoding GCSF-L8-GCSFrEC (1-3) expressed in E.
coli: contains GCSF linked via G4Sx8 to GCSF extracellular receptor domains 1-3 (Ig, BN
and BC). *refers to stop codon; Figure 6b amino acid sequence length = 529aa (not
including Methionine start)
Figure 7a nucleic acid encoding GCSF-L6-GCSFrEC (1-2): contains GCSF linked via
G4Sx6 to GCSF extracellular receptor domains 1-2 (Ig and BN). *refers to stop codon.
Signal sequence in bold and lower case; Figure 7b amino acid sequence length = 404aa
(not including signal sequence)
Figure 8a nucleic acid encoding GCSF-L6-GCSFrEC (2-3): contains GCSF linked via
G4Sx6 to GCSF extracellular receptor domains 2-3 (BN and BC). *refers to stop codon.
Signal sequence in bold and lower case; Figure 8b amino acid sequence length = 416aa
(not including signal sequence)
Figure 9a nucleic acid encoding GCSFrEC (1-3)-L6-GCSF: contains GCSFrEC (domains
1-3) linked via G4Sx6 to GCSF. "refers to stop codon. Signal sequence in bold and lower
case; Figure 9b amino acid sequence length = 511aa (not including signal sequence)
Figure 10a nucleic acid encoding GCSFrEC (2-3)-L6-GCSF expressed in a mammalian
cell line: contains GCSFrEC (domains 2-3) linked via G4Sx6 to GCSF. *refers to stop
codon. Signal sequence in bold and lower case; Figure 10b amino acid sequence length
= 416aa (not including signal sequence)
Figure 11a nucleic acid sequence encoding GCSFrEC expressed in a mammalian cell
line: contains GCSF receptor extracellular domains 1-3. Signal sequence in is bold and
lower case. * refers to stop codon; Figure 11b amino acid sequence length = 307aa (not
including signal sequence); and
Figure 12a nucleic acid sequence GCSFrEC (Extracellular domains 1-3) expressed in
pET21a (+) with 6 x histidine tag (*refers to stop codon, letters in bold refer to extra Xho1
restriction site and 6x Hist-tag); Figure 12b amino acid sequence length = 315aa (not
including Met);

Figure 13 a) PCR was used to generate DNA consisting of the gene of interest flanked
by suitable restriction sites (contained within primers R1-4). b) The PCR products were
ligated into a suitable vector either side of the linker region, c) The construct was then
modified to introduce the correct linker, which did not contain any unwanted sequence
(i.e. the non-native restriction sites); and
Figure 14 a) Oligonucleotides were designed to form partially double-stranded regions
with unique overlaps and, when annealed and processed would encode the linker with
flanking regions which would anneal to the ligand and receptor, b) PCRs were performed
using the "megaprimer" and terminal primers (R1 and R2) to produce the LR-fusion gene.
The R1 and R2 primers were designed so as to introduce useful flanking restriction sites
for ligation into the target vector;
Figure 15 is the complete amino acid sequence of granulocyte colony stimulating factor
receptor;
Figure 16 illustrates western blot analysis of CHO flpln stable cell lines expressing GCSF
chimeric constructs Lane 1= 4A1; Lane 2 = 4D1; Lane 3 = Mock media; Lane 4 = 4A1;
Lane 5 = 4D1; lane 6 = Mock media; A = Non reducing conditions; B = reducing
conditions. Both 4A1 and 4D1 run between 75 and 100 kDa. GCSF runs between 37 and
45 kDa as expected for the glycosylated protein;
Figure 17 is a schematic diagram of the GCSF LR-fusion constructs;
Figure 18 is an immuno-blot analysis of CHO Flp-ln stable cell lines expressing 4A1 and
4D1 constructs. Lane M = Markers (at 250, 150, 100, 75, 50, 37, 25 and 20kDa); Lane 1 =
4A1; Lane 2 = 4D1; Lane 3 = Mock media; Lane 4 = 4A1; Lane 5 = 4D1; lane 6 = Mock
media. Lanes 1-3 = reducing conditions and lanes 4-6 = non reducing conditions;
Figure 19 is an immuno-blot analysis of CHO Flp-ln stable cell lines expressing GCSF,
4B1, 4C1, 4C2 and 4E1 constructs. Lane M = Markers (at 250, 150, 100, 75, 50, 37, 25,
20 and 15kDa); - = no DTT (non-reduced); + = with DTT (reduced);
Figure 20 (A) Coomassie stained gels of the 4A1 purification steps. Lane M = Markers (at
250, 150, 100, 75, 50, 37, 25, 20 and 15kDa); Lane 1 = Crude media 10x concentrate;

Lane 2 = pH4.5 precipitation pellet, Lane 3 = pH 4.5 precipitation supernatant, Lane 4 =
pH 5.5 SP unbound, Lane 5 = pH 5.5 SP bound, Lane 6-8 = fractions 4 to 6 from the pH
8.0 Q column, Lane 9 = 50% ammonium sulphate precipitation pellet. (B) Immuno-blot of
the purified 4A1. Lane M = Markers (as above), Lane 1 = 4A1 (with DTT; reduced), Lane
2 = 4A1 (no DTT; non-reduced); and
Figure 21 illustrates an in vitro bioassay measuring activity of GCSF, Neulasta and the
GCSFLR fusion 4A1.
Materials and Methods
In vitro testing
In vitro methods to test the activity of the GCSF fusion polypeptides are known in the art.
For example, it is known to harvest blood, bone and spleen cells from an animal to test
the colony forming ability of GCSF (see Liu et al Blood, 15 May 2000; 95(10), p3025-
3031). In addition, the use of cells that express GCSFR for example M-NFS-60 cells and
that are stimulated to proliferate as measured by 3H- thymidine is known.
In vivo testing
Various animal models are available to test the activity of GCSF. For example, Harada et
al (Nature Medicine 11: 305-311, 2005) describes a mouse model of myocardial
infarction in which the effects of GCSF were tested to monitor the effects of administered
recombinant GCSF on cardiac function.
Immunological testing
Immunoassays that measure the binding of granulocyte colony stimulating factor to
polyclonal and monoclonal antibodies are known in the art. Commercially available
granulocyte colony stimulating factor antibodies are available to detect granulocyte
colony stimulating factor in samples and also for use in competitive inhibition studies. For
example see, http://www.scbt.com/index.html, Santa Cruz Biotechnology Inc.

Recombinant Production of fusion proteins
The components of the fusion proteins were generated by PCR using primers designed
to anneal to the ligand or receptor and to introduce suitable restriction sites for cloning
into the target vector (Fig 13a). The template for the PCR comprised the target gene and
was obtained from IMAGE clones, cDNA libraries or from custom synthesised genes.
Once the ligand and receptor genes with the appropriate flanking restriction sites had
been synthesised, these were then ligated either side of the linker region in the target
vector (Fig 13b). The construct was then modified to contain the correct linker without
flanking restriction sites by the insertion of a custom synthesised length of DNA between
two unique restriction sites either side of the linker region, by mutation of the linker region
by ssDNA modification techniques, by insertion of a primer duplex/multiplex between
suitable restriction sites or by PCR modification (Fig13c).
Alternatively, the linker with flanking sequence, designed to anneal to the ligand or
receptor domains of choice, was initially synthesised by creating an oligonucleotide
duplex and this processed to generate double-stranded DNA (Fig 14a). PCRs were then
performed using the linker sequence as a "megaprimer", primers designed against the
opposite ends of the ligand and receptor to which the "megaprimer" anneals to and with
the ligand and receptor as the templates. The terminal primers were designed with
suitable restriction sites for ligation into the expression vector of choice (Fig 14b).
Expression and Purification of Fusion Proteins
Expression was carried out in a suitable system (e.g. mammalian CHO cells, E. coli,) and
this was dependant on the vector into which the LR-fusion gene was generated.
Expression was then analysed using a variety of methods which could include one or
more of SDS-PAGE, Native PAGE, western blotting, ELISA.
Once a suitable level of expression was achieved the LR-fusions were expressed at a
larger scale to produce enough protein for purification and subsequent analysis.
Purification was carried out using a suitable combination of one or more chromatographic
procedures such as ion exchange chromatography, hydrophobic interaction
chromatography, ammonium sulphate precipitation, gel filtration, size exclusion and/or

affinity chromatography (using nickel/cobalt-resin, antibody-immobilised resin and/or
ligand/receptor-immobilised resin).
Purified protein was analysed using a variety of methods which could include one or
more of Bradford's assay, SDS-PAGE, Native PAGE, western blotting, ELISA.
Characterisation of LR-fusions
Denaturing PAGE, native PAGE gels and western blotting were used to analyse the
fusion polypeptides and western blotting performed with antibodies non-conformationally
sensitive to the LR-fusion. Native solution state molecular weight information can be
obtained from techniques such as size exclusion chromatography using a Superose
G200 analytical column and analytical ultracentrifugation.
Statistics
Two groups were compared with a Student's test if their variance was normally
distributed or by a Student-Satterthwaite's test if not normally distributed. Distribution was
tested with an F test. One-way ANOVA was used to compare the means of 3 or more
groups and if the level of significance was p<0.05 individual comparisons were performed
with Dunnett's tests. All statistical tests were two-sided at the 5% level of significance and
no imputation was made for missing values.
Construction of chimeric clones
GCSF extracellular receptor domains 1-3 were PCR'd directly from a clone obtained from
the Image Consortium and cloned into the mammalian expression vector pSecTag-link.
Both genes for 4A1 (Figure 3a; Figure 3b) and 4D1 (Figure 9a; Figure 9b) were
constructed using gene synthesis and cloned into the mammalian expression vector
pSecTag-link to form pGCSFsecTag-4A1 and 4A5
Mammalian stable expression of GCSF and Chimeric clones
A mammalian expression system has been established using a modified Invitrogen
vector pSecTag-V5/FRT-Hist. This system allows for the rapid generation of stable
clones into specific sites within the host genome for high expression. This can be used
with either secreted or cytoplasmic expressed proteins. Flp-ln host cell lines (flp-ln CHO)

have a single Flp recombinase target (FRT) site located at a transcriptionally active
genomic locus. Stable cell lines are generated by co-transfection of vector (Containing
FRT target site) and pOG44 (a [plasmid that transiently expresses flp recombinase) into
Flp-ln cell line. Selection is with Hygromycin B. There is no need for clonal selection
since integration of DNA is directed. Culturing Flp-ln Cell lines: followed manufactures
instruction using basic cell culture techniques.
Stable transfection of CHO Flp-ln cells using Fugene-6
The day before transfection CHO Flp-ln cells were seeded at 6 x 10E5 per 100mm petri
dish in a total volume of 10ml of Hams F12 media containing 10% (v/v) Fetal Calf
Serum, 1% Penicillin/streptomycin and 4mM L-glutamine. The next day added 570 µl of
serum free media (containing no antibiotics) to a 1.5ml polypropylene tube. 30µl of
fugene-6 was then added and mixed by gentle rolling. A separate mix of plasmids was
set up for each transfection which combined 2ug plasmid of interest with 18µg pOG44
(plasmid contains recombinase enzyme necessary for correct integration of plasmid into
host genome). Control plate received no plasmid. This was mixed with fugene-6 by gentle
rolling, left @ RmT for 15 minutes, then applied drop-wise to the surface of the each petri
dish containing CHO Flp-ln cells in F12 media + 10% FCS. The plates were gently rolled
to ensure good mixing and left for 24 hrs @ 37°C/5% C02. The next day media was
exchanged for selective media containing hygromycin B @ 600ug/ml. Cells were
routinely kept at 60% confluency or less. Cells were left to grow in the presence of
600ug/ml hygromycin B until control plate cells (non transfected cells) had died (i.e. no
hygromycin resistance).
SDS-PAGE Analysis and Western blotting
Stable transfected CHO Flp-ln cell lines were grown in 75cm2 flasks for approximately 3-
4 days, at which point samples were taken for analysis. Samples were mixed with an
equal volume of Laemmli loading buffer in the presence and absence of 25mM DTT and
boiled for 5 minutes. Samples were separated on a 4-20% (w/v) bis-acrylamide gel and
transferred to a PVDF membrane (Figure 16). After blocking in 5% (w/v) Milk protein in
PBS-0.05% (v/v) Tween 20, sample detection was carried out using a specific anti-
GCSF antibody together with a Horse Radish Peroxidase (HRP) conjugated secondary
antibody. Visualisation was by chemiluminesence on photographic film using an HRP
detection kit.

Construction of LR-fusions
4A1 and 4D1 were gene synthesised (Genecust, France) and inserted into the
mammalian expression vector pSegTag. 4B1 and 4E1 were generated by using PCR to
truncate the 4A1 and 4D1 genes, respectively. 4A2, 4C1 and 4C2 were generated by
synthesising a primer duplex for the linker region and using PCR to extend this into the
GCSF and GCSFR sequences. 4A2 was not synthesised due to the failure of the PCRs
to extend the (G4S)8 linker sequence into the full length gene. 4C2 was generated as a
by-product of the synthesis of 4C1. A schematic of constructs for GCSF-LR fusion
protein is shown in Figure 17.
Expression of LR-fusions
A mammalian expression system has been established using a modified Invitrogen
vector pSecTag-V5/FRT-Hist. This vector is used in Invitrogen's Flp-ln system to direct
integration of the target gene into the host cell line, allowing rapid generation of stable
clones into specific sites within the host genome for high expression.
Culturing Flp-ln Cell lines: followed manufactures instruction using basic cell culture
techniques.
Stable cell lines were generated in 6-well plates using Fugene-6 as the transfection
reagent. The CHO Flp-ln cells were co-transfected with the expression vector and
pOG44, a plasmid that expresses flp recombinase an enzyme which causes the
recombination of the LR-fusion gene into a "hot-spot" of the cell chromosome.
Hygromycin B was used to select for cells with positive recombinants.
Once the stable cell lines had been established they were grown on 75cm2 culture plates,
at a confluency of 50-70% the media was changed to serum free media. The cultures
were incubated for a further 2-4 days after which media samples were taken. These were
run on 13% SDS-PAGE gels and transferred to PVDF membrane for immuno-blotting.
After blocking in 5% (w/v) milk protein in PBS + 0.05% (v/v) Tween 20, sample detection
was carried out using a specific anti-GCSF antibody together with a Horse Radish
Peroxidase (HRP) conjugated secondary antibody. Visualisation was by

chemiluminesence on photographic film using an HRP detection kit. The immuno-blots
showing the expression of the LR-fusions are shown in Figures 18 and 19.
Purification of LR-fusions
The purification methodology for GCSF-LR is detailed below and shown in Figure 20.
a) Protease inhibitors were added to a 10x concentrated media from cells
expressing 4A1.
b) The 10x concentrated media was dialysed against 50mM TRIS, 1mM EDTA, pH
8.0 for 2-4 hours.
c) The protein and dialysis tubing from (2) was transferred to a solution of 10mM
TRIS, 1mM EDTA, pH 8.0 for 16 hours (overnight).
d) 0.25 volumes of 0.1M acetate buffer, pH 4.5 was added. The pH was checked
using pH indicator strips and more 0.1M acetate buffer, pH 4.5 added in 0.1ml
aliquots until the pH reached 4.5.
e) This was then incubated on ice for 2 hours with periodic mixing.
f) Centrifugation was performed to remove the precipitate and the supernatant
transferred to a new tube.
g) 0.5M / 0.1M NaOH was added to the supernatant in 0.1ml aliquots until the pH
changed to 5.5 - analysed pH using pH indicator strips.
h) The solution was then loaded onto a 5ml SP FF column pre-equilibrated with
25mM acetate buffer, pH 5.5. The unbound protein was collected,
i) The unbound fraction was dialysed against 25mM TRIS, 1mM EDTA, pH 8.0 for
16 hours (overnight),
j) The dialysed sample was loaded onto a 5ml Q FF column pre-equilibrated with
25mM TRIS, pH 8.0.
k) The column was washed with 20 column volumes of 25mM TRIS, pH 8.0.
I) Protein was then eluted off the column using a 0-1M NaCI gradient over 20
column volumes, collecting 1 column volume fractions. [ 4A1 is eluted in fraction
4-7 on a 5ml column ].
m) Fractions containing >70% pure 4A1 were pooled and incubated on ice.
n) An equal volume of ice cold saturated ammonium sulphate solution was added to
give 50% ammonium sulphate saturation. Incubated on ice for 2-3 hours,
o) The protein sample was then centrifuged to pellet precipitated protein,
p) The pellet was re-suspended in PBS and dialysed against PBS.
q) The protein sample was then analysed.

In vitro Bioassay
Cell Preparation
AML-193 cells (ATCC, Batch No. 3475266) were removed from liquid nitrogen storage
and placed into a 37°C waterbath for 2 min. The contents of the vial were then
transferred to a 15 ml tube containing 9 ml of culture medium (5% FBS, 4mM L-
glutamine, 100 U/ml penicillin, 100 ug/ml streptomycin, 5 ng/ml GM-CSF, 5 ug/ml insulin,
5 ug/ml transferrin in Iscove's modified Dulbecco's medium). Cells were centrifuged for 5
min at 123xg, the cell pellet was resuspended in culture medium and cell density
adjusted to 2.3x105 cells/ml.
Cell culture
Cells were cultured in CO2 incubator (5% CO2, 37°C) in culture medium at a density of
3x105 - 2x106 cells/ml. Passages were performed twice a week ensuring cell density did
not exceed 2.5x106 cells/ml. Cell viability was assessed by trypan blue exclusion. Prior to
assay cells were washed 3 times with PBS by spinning for 5 min at ~125xg. The pellet
was then reconstituted in assay medium (5% FBS, 4mM L-glutamine, 100 U/ml penicillin,
100 ug/ml streptomycin, 5 ug/ml insulin, 5 ug/ml transferrin in Iscove's modified
Dulbecco's medium) and cell density was adjusted to 5x105 cells/ml.
Standard/sample preparation
Appropriate dilutions of GCSF protein solution of 4A1 were made in PBS (1% BSA) for
bioactivity testing. GCSF (international standard, NIBSC, Batch No 88/502) were
reconstituted in 50 % solution of phosphate buffered saline and water (both sterile) to a
concentration of 10 ng/ml (10000 Ill/ml), divided into 40 pi aliquots and stored at -80°C.
On each day of assay 1 vial was removed from the freezer and working concentrations
were prepared.
The in vitro bioactivity for GCSF-LR (4A1) is shown in Figure 21.

0772P.WO
Cys Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu Gin
20 25 30
Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu val
35 40 45
Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu ser Ser cys
50 55 60
Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser
65 70 75 80
Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie Ser
85 90 95
Pro Glu Leu Gly pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp
100 105 110
Phe Ala Thr Thr lie Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro
115 120 125
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe
130 135 140
Gin Arg Arg Ala Gly Gly val Leu Val Ala Ser His Leu Gin Ser Phe
145 150 155 160
Leu Glu Val Ser Tyr Arg val Leu Arg His Leu Ala Gin Pro Gly Gly
165 170 175
Gly Gly ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
180 185 190
Gly ser Gly Gly Gly Gly ser Gly Gly Gly Gly Ser Glu cys Gly His
195 200 205
lie Ser val Ser Ala Pro lie Val His Leu Gly Asp Pro lie Thr Ala
210 215 220
Ser cys lie lie Lys Gin Asn Cys Ser His Leu Asp Pro Glu Pro Gin
225 230 235 240
lie Leu Trp Arg Leu Gly Ala Glu Leu Gin Pro Gly Gly Arg Gin Gin
245 250 255
Page 9

iv) a nucleic acid sequence as represented in SEQ ID NO: 17;
v) a nucleic acid sequence as represented in SEQ ID NO: 19; or
a nucleic acid molecule comprising a nucleic sequence that hybridizes under stringent
hybridization conditions to SEQ ID NO:5-SEQ ID NO: 19 and which encodes a
polypeptide that has granulocyte colony stimulating factor receptor modulating activity.
18 A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
encodes a polypeptide that has agonist activity.
19. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
encodes a polypeptide that has antagonist activity.
20. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 5.
21. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 7.
22. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 9.
23. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 11.
24. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 13.
25. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises of a nucleic acid sequence as represented in SEQ ID NO: 15.
26. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 17.
27. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 19.

28. A fusion polypeptide according to any of claims 2-9 wherein granulocyte colony
stimulating factor polypeptide is linked to the cytokine binding domain wherein said
granulocyte colony stimulating factor polypeptide is positioned amino terminal to said
cytokine binding domain in said fusion polypeptide.
29. A fusion polypeptide according to any of claims 2-9 wherein granulocyte colony
stimulating factor polypeptide is linked to the cytokine binding domain wherein said
granulocyte colony stimulating factor polypeptide is positioned carboxyl-terminal to said
cytokine binding domain in said fusion polypeptide.
30. A fusion polypeptide according to any of claims 2-11 wherein granulocyte colony
stimulating factor polypeptide is linked to the binding domain of the granulocyte colony
stimulating factor receptor polypeptide by a peptide linker.
31. A fusion polypeptide according to claim 12 wherein said peptide linking molecule
comprises at least one copy of the peptide Gly Gly Gly Gly Ser.
32. A fusion polypeptide according to claim 13 wherein said peptide linking molecule
comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the peptide Gly Gly Gly Gly Ser.
33. A fusion polypeptide according to claim 14 wherein said peptide linking molecule
consists of 6 copies of the peptide Gly Gly Gly Gly Ser.
34. A fusion polypeptide according to any of claims 2-11 wherein said polypeptide
does not comprise a peptide linking molecule and is a direct fusion of granulocyte colony
stimulating factor polypeptide and the binding domain of granulocyte colony stimulating
factor receptor polypeptide.
35. A nucleic acid molecule comprising a nucleic acid sequence selected from:


WE CLAIM :
1. A nucleic acid molecule comprising a nucleic acid sequence that encodes a
polypeptide having the activity of granulocyte colony stimulating factor comprising a
granulocyte colony stimulating factor polypeptide linked, directly or indirectly, to at least
one cytokine binding domain of the granulocyte colony stimulating factor receptor
polypeptide.
2. A fusion polypeptide comprising: the amino acid sequence of granulocyte colony
stimulating factor polypeptide, or active part thereof linked, directly or indirectly, to at
least one cytokine binding domain of the granulocyte colony stimulating factor receptor
polypeptide.
3. A fusion polypeptide according to claim 2 wherein said fusion polypeptide
comprises two cytokine binding domains of the granulocyte colony stimulating factor
receptor polypeptide.
4. A fusion polypeptide according to claim 2 or 3 wherein said polypeptide further
comprises an immunoglobulin-like domain.
5. A fusion polypeptide according to any of claims 2-4 wherein said polypeptide
includes at least one fibronectin III domain.
6. A fusion polypeptide according to any of claims 2-5 wherein said polypeptide
comprises amino acid residues 97-201 as represented in SEQ ID NO: 31.
7. A fusion polypeptide according to any of claims 2-6 wherein said polypeptide
comprises amino acid residues 202-313 of SEQ ID NO: 31.
8. A fusion polypeptide according to any of claims 2-7 wherein said polypeptide
comprises amino acid residues 97-313 of SEQ ID NO: 31.
9. A fusion polypeptide according to any of claims 2-8 wherein said polypeptide
comprises amino acid residues 1-97 of SEQ ID NO: 31.

10. A fusion polypeptide according to any of claims 2-9 wherein granulocyte colony
stimulating factor polypeptide is linked to the cytokine binding domain wherein said
granulocyte colony stimulating factor polypeptide is positioned amino terminal to said
cytokine binding domain in said fusion polypeptide.
11. A fusion polypeptide according to any of claims 2-9 wherein granulocyte colony
stimulating factor polypeptide is linked to the cytokine binding domain wherein said
granulocyte colony stimulating factor polypeptide is positioned carboxyl-terminal to said
cytokine binding domain in said fusion polypeptide.
12. A fusion polypeptide according to any of claims 2-11 wherein granulocyte colony
stimulating factor polypeptide is linked to the binding domain of the granulocyte colony
stimulating factor receptor polypeptide by a peptide linker.
13. A fusion polypeptide according to claim 12 wherein said peptide linking molecule
comprises at least one copy of the peptide Gly Gly Gly Gly Ser.
14. A fusion polypeptide according to claim 13 wherein said peptide linking molecule
comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the peptide Gly Gly Gly Gly Ser.
15. A fusion polypeptide according to claim 14 wherein said peptide linking molecule
consists of 6 copies of the peptide Gly Gly Gly Gly Ser.
16. A fusion polypeptide according to any of claims 2-11 wherein said polypeptide
does not comprise a peptide linking molecule and is a direct fusion of granulocyte colony
stimulating factor polypeptide and the binding domain of granulocyte colony stimulating
factor receptor polypeptide.
17. A nucleic acid molecule comprising a nucleic acid sequence selected from:

iv) a nucleic acid sequence as represented in SEQ ID NO: 17;
v) a nucleic acid sequence as represented in SEQ ID NO: 19; or
a nucleic acid molecule comprising a nucleic sequence that hybridizes under stringent
hybridization conditions to SEQ ID NO:5-SEQ ID NO: 19 and which encodes a
polypeptide that has granulocyte colony stimulating factor receptor modulating activity.
18 A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
encodes a polypeptide that has agonist activity.
19. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
encodes a polypeptide that has antagonist activity.
20. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 5.
21. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 7.
22. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 9.
23. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 11.
24. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 13.
25. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises of a nucleic acid sequence as represented in SEQ ID NO: 15.
26. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 17.
27. A nucleic acid molecule according to claim 17 wherein said nucleic acid molecule
comprises a nucleic acid sequence as represented in SEQ ID NO: 19.

28. A polypeptide encoded by the nucleic acid according to any of claims 17-27
29. A polypeptide comprising an amino acid sequence selected from the group
consisting of: SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 25, 26, 27, 28, 29 or 30.
30. A polypeptide according to claim 29 wherein said polypeptide has agonist activity.
31. A polypeptide according to claim 29 wherein said polypeptide has antagonist
activity.
32. A homodimer consisting of two polypeptides wherein each of said polypeptides
comprises:
i) a first part comprising granulocyte colony stimulating factor, or a receptor
binding domain thereof, optionally linked by a peptide linking molecule to
ii) a second part comprising the cytokine homology binding domain or part
thereof, of the granulocyte colony stimulating factor receptor.
33. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising of SEQ ID NO: 6.
34. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising of SEQ ID NO: 8.
35. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 10.
36. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 12.
37. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 14.
38. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 16.

39. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 18.
40. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 20.
41. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising or consisting of SEQ ID NO: 25.
42. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 26.
43. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 27.
44. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 28.
45. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 29.
46. A homodimer according to claim 32 wherein said homodimer comprises two
polypeptides comprising SEQ ID NO: 30.
47. A vector comprising a nucleic acid molecule according to any of claims 1 or 17-
27.
48. A cell transfected or transformed with a nucleic acid molecule according to any of
claims 1 or 17-27 or vector according to claim 47.
49. An isolated cell according to claim 48 wherein said cell is a eukaryotic cell.
50. A cell according to claim 48 wherein said cell is a prokaryotic cell.

t
51. A pharmaceutical composition comprising a polypeptide according to any of
claims 2-16 or 28-31 including an excipient or carrier.
52. A composition according to claim 51 wherein said pharmaceutical composition is
combined with a further therapeutic agent.

We disclose granulocyte colony stimulating factor fusion polypeptides; nucleic acid
molecules encoding said polypeptides and methods of treatment that use said proteins.