ES2368092T3 - CSF3R POLIPEPTIDES AND USES OF THE SAME. - Google Patents

Abstract

A variant of the soluble isolated polypeptide of the CSF3R, wherein said polypeptide variant comprises or consists of a sequence of a ligand binding domain, lacks a transmembrane functional domain and lacks a cytoplasmic domain, where said ligand binding domain is a type domain immunoglobulin of the CSF3R, wherein said polypeptides further include following the immunoglobulin type domain amino acid residues 97-165 of SEQ ID NO: 4 or a fragment of at least 50 amino acids of amino acid residues 97-165 of SEQ ID NO : 4 and where said polypeptides retain the ability to bind the natural ligands of CSF3R and are further characterized in that the variant of the isolated soluble polypeptide of CSF3R is selected from SEQ ID NO: 2, a mature form of SEQ ID NO: 2 , a glycosylated form of SEQ: ID NO: 2, a pegylated form of SEQ ID NO: 2 or a functional equivalent of SEQ ID NO: 2, where the functional equivalent It has at least more than 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 2

Description

CSF3R polypeptides and uses thereof

The present invention relates to polypeptides of CSF3R. The invention also relates to nucleic acids encoding said polypeptides, vectors comprising said nucleic acids and recombinant cells containing them, as well as the corresponding pharmaceutical compositions. The invention further discloses methods for producing said polypeptides.

BACKGROUND

Granulocyte colony stimulating factor (GCSF or CSF3) exerts its function by activating a membrane receptor, colony stimulating factor receptor 3 (CSF3R), which belongs to the super family of hematopoietin receptors, to the which is also referred to as class I cytokine receptors. CSF3R is also known as the granulocyte colony stimulating factor (GCSFR) or CD114 receptor.

It has been found that various lymphokine receptors, hematopoietic growth factors and growth hormone related molecules share a common binding domain. These receptors are known as hematopoietin receptors and the corresponding ligands as hematopoietin. Likewise, hematopoietins were subdivided into two large structural groups: large / long and small / short hematopoietins. A subset of individual receptor chains that are part of the receptor complexes for large hematopoietins contain common structural elements in their extracellular parts: an immunoglobin-like domain, a hematopoietin receptor domain and 3 type III fibronectin domains (2 in the leptin receptor). This subgroup was called the "gp130 receptor family" (Mosley, et al. J. Biol. Chem. 1996, 271, 32635-43) and includes the Leptin receptor (LPTR), the granulocyte colony stimulating factor receptor ( GCSFR), the common beta chain of Interleukin-6 / -11 / LIF / OSM / CNTF (GP130), the leukemia inhibitor factor receptor (LIFR), the Oncostatin-M receptor beta chain (OSMR), the Interleukin-12 receptor beta-1 chain (IL12RB1), Interleukin-12 receptor beta-2 chain (IL12RB2). These receptor chains are homodimerized (GCSFR, GP130, LPTR) or heterodimerized (GP130 with LIFR or OSMR, IL12RB1 with IL12RB2) after binding to the cognate cytokine.

The human CSF3R protein is an 826 amino acid polypeptide comprising a signal peptide (amino acid residues 1-24), an extracellular domain located in the N-terminal region of the protein (amino acid residues 25-627), an intracellular domain ( amino acid residues 651-836) and a transmembrane region (amino acid residues 628-650). The extracellular portion contains an N-terminal immunoglobulin (Ig) type domain (amino acid residues 25 to 117), a cytokine receptor homology domain (CRH) (amino acid residues 122 to 330) and 3 fibronectin domains type III ( FNIII). Within the CRH region are 2 FNIII domains, 4 conserved cysteine residues and a WSXWS conserved motif (amino acid residues 318 to 322) that stabilizes the CRH domain. Ig and CRH domains are critical for high affinity binding. In the intracellular domain there is a box motif 1 (amino acid residues 658 to 666) necessary for the interaction and / or activation of JAK.

In Hiratoka et al. (J. Biol. Chem. 1995; 270: 25928-25934) a soluble CSF3R is described consisting of the lg-like domain and the aminoterminal part of the CRH domain.

GCSF stimulates the proliferation, survival and maturation of cells compromised with the neutrophil granulocyte lineage by binding to the specific GCSF receptor (see Hartung T., et al., Curr. Opin. Hematol. 1998; 5: 221-5 ). The CSF3R receptor does not possess intrinsic kinase activity in the cytoplasmic domain and uses intracellular protein kinases associated with the receptor such as Janus tyrosine kinase (JAK) to initiate signal transduction. Stimulation of cells with G-CSF has been shown to activate multiple signal transduction pathways, including signal transducers and transcription activators (STAT), Ras / Raf / Erk, phosphatidylinositol 3-kinase (PI3-K ) / Akt, and also Stat proteases such as Stat5 protease. Unregulated activation of Stat5 can lead to aberrant cellular responses to CSF3 and contribute to leukemogenesis.

GCSF is generally used for the treatment of different types of neutropenia in humans. It is one of the few growth factors approved for clinical use. In particular, it is used to reduce chemotherapy-induced cytopenia (Viens et al., J. de Clin. Oncology, Vol. 20, No. 1, 2002: 24-36). GCSF has also been used therapeutically for infectious diseases as a potential adjuvant agent (Hubel et al., J. de Infectious Diseases, Vol. 185: 1490-501, 2002).

In a mouse myocardial infarction model, treatment with CSF3 did not affect the initial magnitude of the infarction on day 3 but improved cardiac function only 1 week after the infarction and the beneficial effects were reduced by the delay in the beginning of treatment (Harada et al. Nature Med. Vol. 11: 305-311, 2005). CSF3 induced antiapoptotic proteins and inhibited apoptotic cardiomyocyte death. Likewise, CSF3 reduced apoptosis of endothelial cells and increased vascularization in hearts that had suffered heart attacks. In Harada et al. It was suggested that CSF3 promotes the survival of cardiac myocytes and prevents remodeling of the left ventricle after myocardial infarction.

The application of a unit dose of G-CSF in patients suffering from community-acquired pneumonia (CAP) increased to the duration of survival and activation of neutrophils, combined with a sustained release of anti-inflammatory cytokines (Droemann et al. Respiration 2005 Dec. 12. Epub prior to publication).

CSF3 is also beneficial for the prevention and / or treatment of immunological diseases, for example, graft versus host disease, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease and diabetes (Rutella et al. J. Immunol. 2005 Dec. 1 ; 175 (11): 7085-91).

SUMMARY OF THE INVENTION

The present invention discloses new variants of CSF3R polypeptides. The invention further discloses nucleic acids encoding said polypeptides, vectors comprising said nucleic acids, in particular, expression vectors and recombinant cells containing them, as well as the corresponding pharmaceutical compositions. The invention further discloses methods for producing said polypeptides.

More particularly, the invention results from the identification, isolation and characterization of a new, naturally occurring soluble splice variant of human CSF3, called sCSF3R, which has structural and biological properties, which represents a valuable pharmaceutical product.

An object of the present invention thus lies in variants of the polypeptide isolated from CSF3R. The polypeptide variants of the present invention comprise the sequence of a CSF3 ligand binding domain, lack a transmembrane functional domain and lack a cytoplasmic domain, where said ligand binding domain is an immunoglobulin type domain of CSF3R, where said polypeptides further include after the immunoglobulin type domain amino acid residues 97-165 of SEQ ID NO: 4, or a fragment of at least 50 amino acids of amino acid residues 97-165 of SEQ ID No.4, and wherein said polypeptides retain the ability to bind the natural ligands of CSF3R, further characterized in that the variant of isolated soluble CSF3R polypeptides is selected from SEQ ID NO: 2, a mature form of SEQ ID NO: 2, a glycosylated form of the SEQ ID NO: 2, a pegylated form of SEQ ID NO: 2 or a functional equivalent of SEQ ID NO: 2, where the functional equivalent has at least more than 85%, 90%, 95%, 98% or 99% d and identity with SEQ ID NO: 2. The CSF3R polypeptides of the present invention represent soluble forms of CSF3R that can be used as agonists or antagonists of these in various pathological conditions.

Another object of the present invention lies in the mature form of the CSF3R as defined above.

Another object of the present invention lies in a fusion protein comprising a variant of CSF3R polypeptide as defined above.

Another object of the present invention lies in a conjugate comprising a variant of CSF3R polypeptide as defined above.

A further object of the present invention lies in a nucleic acid encoding a polypeptide variant of the CSF3R or a fusion protein as defined above, as well as any cloning or expression vector comprising said nucleic acid.

The invention further relates to recombinant host cells comprising a vector or nucleic acid as defined above, as well as methods for producing a variant of CSF3R polypeptide as defined above through the use of recombinant cells.

A further aspect of the present invention lies in a pharmaceutical composition comprising a product (for example, a polypeptide, a fusion protein, a conjugate, a receptor complex, a nucleic acid, a vector or recombinant cell) as defined above.

The above products and pharmaceutical composition are particularly suitable, for example, to treat graft versus host disease, cancer, neutropenia, neurological disorders or conditions, pneumonia, autoimmune diseases, hematological diseases, hemopoietic disorders, infectious diseases, inflammatory bowel disease and diabetes. .

LEGENDS OF THE FIGURES

Fig. 1: Protein and DNA sequence of sCSF3R. The position of the PCR primers used for cloning is indicated by arrows.

Fig. 2: Nucleotide sequence with translation of the sCSF3R PCR product.

Fig. 3: Alignment between sCSF3R and membrane bound protein. The membrane-bound protein contains a signal peptide (framed) of amino acids 1 to 24, an Ig C2 type domain (light gray) and 5 fibronectin III domains (dark gray). The variant contains the signal peptide, the Ig-like domain and does not have any fibronectin III domain. The reason for box 1 is necessary for the interaction and / or activation of JAK. The expected molecular weight of sCSF3R is 20667.7761.

DETAILED DESCRIPTION OF THE INVENTION

The present invention results from the identification and characterization of the new biologically active human polypeptide variants of CSF3R. These polypeptides generally comprise the sequence of a mature human CSF3R polypeptide that lacks a transmembrane functional domain. In particular, the present invention lies in the identification of a splicing variant of natural origin of the CSF3R, called sCFS3R (SEQ ID NO: 2). The present invention may comprise variants of naturally occurring or synthetic CSF3R polypeptides and represents valuable therapeutic molecules. Also, due to the absence of a transmembrane functional domain, the CSF3R polypeptide variants of the present invention are not fixed in the cell membrane and can circulate in biological fluids, particularly blood, plasma, serum, lymph or Similar.

The membrane-bound CSF3R extracellular region (Swissprot accession Q99062; SEQ ID NO: 10) comprises six structural domains: an N-terminal Immunoglobulin (Ig) type domain followed by five fibronectin type III (FNIII) domains. The first two domains of FNIII (called D2 and D3) form the homology module of the cytokine receptor (CRH), which contains four cysteine residues in D2 and a WSXWS motif in D3. These characteristics of the CRH module are retained in members of the class 1 cytokine receptor family. The membrane bound CSF3R consists of 15 coding exons. There are 2 non-coding exons in the 5 ’UTR. The start codon is in exon 3 and the end codon is in exon 15.

The soluble CSF3R variant of the present invention (SEQ ID NO: 2) is encoded by exon 3, exon 4 and genomic prolongation of exon 4 at the 4th intron of the gene. The soluble CSF3R variant has a length of 599 base pairs encoding a 189 amino acid polypeptide. The first exon extends from base 1 to base 84 and the second exon extends from base 85 to base 599. Therefore, the protein sequence of the soluble CSF3R variant is characterized by its N-terminal part of the CSF3R bound to the membrane encoded by exon 3 and exon 4 and its 69 supplementary amino acids that come from prolongation in intron 4.

Accordingly, the CSF3R polypeptide variants of the present invention comprise a functional ligand binding domain, but lack a transmembrane functional domain and also lack a cytoplasmic region. The invention shows that said proteins are produced naturally and represent unexpected variants of CSF3R with unusual structural conformation. In fact, the molecules of the present invention contain 69 additional unique amino acids following the Ig-like domain. The additional 69 amino acids consist of amino acid residues 97 to 165 of SEQ ID NO: 4. The sCFS3R of the present invention is also characterized by the fact that it retains only the Ig type domain (in its entirety) of the binding domain to ligand and lacks the CRH module and all type III fibronectin domains. The polypeptides disclosed herein preferably lack the CRH module and / or one, two, three, four, five or all of the fibronectin type III domains. The Ig type domain is necessary for homodimerization of receptors with CSF3 in solution and the Ig type domain showed binding to CSF3 (Layton et al. J Biol. Chem. 2001 Sep. 28; 276 (39): 36779-87.). Therefore, the soluble CSF3R variant of the present invention as expressed in SEQ ID NO: 4, retains the essential characteristics of the CSF3 ligand binding domain, which consists of the Ig type domain.

The polypeptide variants of the CSF3R disclosed herein lack a transmembrane functional domain, that is, they do not contain a functional domain derived from the CSF3R that allows the fixation of the membrane polypeptide. The absence of a transmembrane functional domain may result from the alteration of any amino acid (e.g., the removal, substitution and / or addition of one or more amino acid residues) in a transmembrane domain of the CSF3R resulting in a non-functional transmembrane domain. In a typical embodiment, the absence of a transmembrane functional domain results from the removal of all or part of the amino acid residues that form the transmembrane domain, preferably the removal of amino acid residues 628 to 650 from SEQ ID NO: 10.

In one aspect, the invention provides a variant of the isolated CSF3R polypeptide, wherein said polypeptide variant consists of a sequence of a ligand binding domain, lacks transmembrane functional domain and lacks cytoplasmic domain, wherein said ligand binding domain it is an immunoglobulin type domain of CSF3R, where said polypeptides further include following the immunoglobulin type domain amino acid residues 97-165 of SEQ ID NO: 4, or a fragment of at least 50 amino acids of amino acid residues 97-165 of SEQ ID No.4, and where said polypeptides retain the ability to bind the natural ligands of CSF3R, and further characterized in that the variant of the isolated soluble polypeptide of CSF3R is selected from SEQ ID NO: 2, a mature form of the SEQ ID NO: 2, a glycosylated form of SEQ ID NO: 2, a pegylated form of SEQ ID NO: 2 or a functional equivalent of SEQ ID NO: 2, where it is equivalent The functional has at least more than 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 2.

Current understanding of the mechanisms of soluble receptors includes their role as inhibitors of their respective membrane-bound receptors through competition for ligands, regulators for decreased expression of membrane-bound receptors, ligand stabilizing proteins and participants in ligand-induced signaling. In Ku et al. It is shown that a soluble form of the G-CSF receptor (called sG-CSFR) can enter into synergy with the steel factor (ligand SF, Kit) or ligand Flt3 / Flk2 (FL) which helps the proliferation of primitive hematopoietic progenitors (Ku et al. Blood. 1996 Dec. 1; 88 (11): 4124-31). In Ku et al. It is indicated that the soluble G-CSF receptor seems to transduce the signals by interacting with their respective membrane-bound receptor.

Thus, variants of the soluble CSF3R polypeptide of the present invention represent membrane-bound ligand stabilizing and / or CSF3R proteins, which promote ligand-induced signaling and act as CSF3R agonists. The soluble receptors of the present invention can stabilize a ligand or membrane bound CSF3R or both a ligand and membrane bound CSF3R. Preferably, the ligand is CSF3. Soluble CSF3R can prevent the degradation of its cognated ligands as shown for soluble growth hormone receptors.

In Asano et al. It is shown that soluble chimeric CSF3R can inhibit the biological activity of CSF3 in the formation of normal bone marrow colony (Asano et al. Cancer Res. 1997 Aug. 15; 57 (16): 3395-7). Likewise, soluble chimeric CSF3R completely inhibited the stimulatory effect of CSF3 on the proliferation of leukemic progenitor cells to form colonies of leukemic blasts and represents a potential therapeutic candidate in a clinical application for acute myeloblastic leukemia. In Iwasaki et al. It was suggested that soluble isoforms of CSF3R may compete with membrane-bound CSF3R in white cells and serve as negative regulators of myelopoiesis (Iwasaki et al. J. Immunol. 1999 Dec. 15; 163 (12): 6907- eleven).

Thus, the variants of the soluble CSF3R polypeptide of the present invention may represent decoys that can bind natural endogenous ligands of the CSF3R and thereby reduce the activities mediated by the CSF3R, acting as CSF3R antagonists. In particular, said variants of the soluble polypeptide of the CSF3R act as regulators for the activities mediated by the CSF3R by a negative dominant mechanism.

Preferably, the variants of the soluble CSF3R polypeptide of the present invention represent ligand-stabilized and / or membrane-bound CSF3R proteins that promote ligand-induced signaling and act as CSF3R agonists.

In Layton et al., By modeling the Ig-like domain, it was shown that the CSF3R domain structure is also found in the closest homologue gp130, which is the chain of shared signal transducer receptors of the interleukin family (IL ) -6 cytokines (Layton et al. J Biol. Chem. 2001 Sep. 28; 276 (39): 3677987.). G-CSF3R and gp130 share a 46% sequence similarity in the extracellular region. Likewise, a similar structural relationship is observed between the 2: 2 tetrameric complexes formed by IL-6 with gp30, or CSF3 with CSF3R. The formation of complexes between IL-6 and its soluble receptor (sIL-6R), which interact with gp130, leads to an increase in nuclear expression and translocation of STAT3, which causes the induction of antiapoptotic genes, such as Bcl- x1 (Atreya R and Neurath M F. Clin Rev Allergy Immunol. 2005 June; 28 (3): 18796.).

A receptor complex comprising a variant of CSF3R polypeptide of the present invention is also disclosed.

A receptor complex comprising a fusion protein comprising a variant of CSF3R polypeptide of the present invention is also disclosed.

A complex of receptors formed by the CSF3, variants of the soluble CSF3R of the present invention and the membrane-bound CSF3R are also disclosed.

A receptor complex formed by variants of the soluble CSF3R of the present invention and the membrane bound CSF3R is also disclosed.

A receptor complex formed by the CSF3 and variants of the soluble CSF3R of the present invention are also disclosed.

Preferably, the receptor complex consists of a 2: 2 tetrameric complex.

The soluble polypeptide variants of the CSF3R of the present invention represent natural agonists and / or antagonists of the CSF3R and can be used as such, or in the form of, for example, a fusion protein, a conjugate or receptor complex to treat graft disease. against host, cancer, neutropenia, neurological disorders or conditions, pneumonia, autoimmune diseases, hematological diseases, hematopoietic disorders, infectious diseases, inflammatory bowel disease and diabetes.

Another object of the present invention lies in a soluble CSF3R comprising an immunoglobulin (Ig) type domain and the CRH module fused with an amino acid sequence comprising or consisting of one or more amino acids encoded by intron 4 of SEQ ID NO :one. Preferably, an object of the present invention resides in a soluble CSF3R comprising the immunoglobulin (Ig) type domain linked to an amino acid sequence comprising or consisting of one or more amino acids encoded by intron 4 of SEQ ID NO: 1. Preferably, the Ig type domain is fused to an amino acid sequence that comprises or consists of 50 or 69 amino acids encoded by intron 4 of SEQ ID NO: 1. Alternatively, the Ig type domain is fused to a sequence. of amino acids comprising or consisting of 50 or 69 amino acids of amino acids 97 to 165 of SEQ ID NO: 4 (mature sCSF3R). Preferably, the Ig type domain is fused with an amino acid sequence consisting of amino acids 97 to 165 of SEQ ID NO: 4.

In another aspect, the invention thus provides a polypeptide variant isolated from the CSF3R of the present invention wherein the polypeptide further includes the amino acid sequence encoded by intron 4 of SEQ ID NO: 1 or further includes a fragment of the sequence of amino acids encoded by intron 4 of SEQ ID NO: 1 of at least 50, 60 or 69 amino acids. Preferably, the isolated CSF3R polypeptide further includes an amino acid sequence consisting of amino acids 97 to 165 of SEQ ID NO: 4.

More particularly, the invention is the result of the identification, isolation and characterization of a new naturally occurring soluble splice variant of the human CSF3R, called sCSF3R (SEQ ID NO: 2), which has particular structural and biological properties, It represents a valuable pharmaceutical product. Therefore, an object of the present invention lies in the soluble CSF3R as expressed in SEQ ID NO: 2, its mature form, its glycosylated form and its functional equivalent. Preferably, the mature form of SEQ ID NO: 2 is SEQ ID NO: 4.

Therefore, another object of the present invention lies in the mature form of the soluble CSF3R variant as expressed in SEQ ID NO: 4. Alternatively, an object of the present invention lies in SEQ ID NO: 2 which lacks the signal peptide. Preferably, the signal peptide extends from amino acids 1 to 25 of SEQ ID NO: 2, or amino acids 1 to 24 of SEQ ID NO: 2, or amino acids 1 to 23 of SEQ ID NO: 2.

Other disclosed objects lie in the cloned sequence as expressed in SEQ ID NO: 6, its mature form, its glycosylated form, its functional equivalent or its characteristic fragment. Preferably, the mature form of SEQ ID NO: 6 is SEQ ID NO: 8.

The mature form of the soluble CSF3R variant is also disclosed as expressed in SEQ ID NO: 8. Alternatively, an object disclosed is SEQ ID NO: 6 that lacks a signal peptide. Preferably, the signal peptide extends from amino acids 1 to 25 of SEQ ID NO: 6, or amino acids 1 to 24 of SEQ ID NO: 6, or amino acids 1 to 23 of SEQ ID NO: 6.

A particular object described herein is variants of an isolated CSF3R polypeptide, or its characteristic fragment, wherein said polypeptide variants comprise a sequence of the CSF3 ligand binding domain and lack a transmembrane functional domain. Preferably, the ligand binding domain of CSF3 consists of amino acid residues 25 to 627 of SEQ ID NO: 10, more preferably they consist of amino acid residues 25 to 618 of SEQ ID NO: 10, even more preferably consists in the Ig type domain and the CRH domain of the membrane bound CSF3R or consists of amino acid residues 25 to 330 of SEQ ID NO: 10. The preferred ligand binding domain of CSF3 consists of the Ig type domain of the CSF3R bound to the membrane, or consists of amino acid residues 25 to 120 of SEQ ID NO: 10 (as expressed in SEQ ID NO: 12), or consists of amino acids 25 to 117 of SEQ ID NO: 10.

Another object described herein lies in the isolated polypeptide variants of the CSF3R or its characteristic fragment, wherein said polypeptide variants comprise a sequence of the CSF3 ligand binding domain, lack transmembrane functional domain and lack cytoplasmic region. Preferably, the transmembrane domain consists of amino acid residues 628 to 650 of SEQ ID NO: 10. Preferably, the cytoplasmic region consists of amino acid residues 651 to 836 of the membrane bound CSF3R (SEQ ID NO: 10).

A polypeptide variant isolated from CSF3R of the present invention is also described, wherein said polypeptide variant isolated from CSF3R lacks amino acids 628 to 650 of SEQ ID NO: 10 and / or lacks amino acids 651 to 836 of SEQ ID NO: 10.

A polypeptide variant isolated from CSF3R encoded by intron 4 of SEQ ID NO: 1, or a fragment thereof, is also described.

The present application describes a polypeptide variant isolated from CSF3R consisting of amino acids 97 to 165 of SEQ ID NO: 4.

An isolated polypeptide variant of CSF3R encoded by intron 4 of SEQ ID NO: 1, or a fragment thereof, fused with one or more amino acids of the ligand binding domain, preferably the Ig-like domain, is also disclosed.

A polypeptide variant isolated from CSF3R is described, wherein said polypeptide comprises or consists of the amino acid sequence encoded by intron 4 of SEQ ID NO: 1 or a fragment of the amino acid sequence encoded by intron 4 of SEQ ID NO: 1 of at least 6, 7, 8, 10, 15, 20, 25, 30, 40, 50, 60 or 69 amino acids. The isolated CSF3R polypeptide can be fused with one or more amino acids of the CSF3R ligand binding domain.

Another object of the present invention lies in the Ig-like domain of CSF3R bound to the membrane fused with amino acid residues 97 to 165 of SEQ ID NO: 4. Another object of the present invention is in the Ig-like domain of bound CSF3R to the membrane or a fragment of this fused with a fragment of the sequence consisting of amino acid residues 97 to 165 of SEQ ID NO: 4 where the fragment has at least 50 amino acids of amino acid residues 97-165 of the SEQ ID NO: 4.

Preferably, the ligand binding domain of the present invention is an immunoglobulin-like domain of CSF3R.

Preferably, the immunoglobulin type domain of the present invention is an Ig type C2 type domain. Preferably, the type C2 type Ig domain contains two cysteine residues that form a disulfide bridge.

Preferably, an Ig type domain of the present invention contains eight β strands. Preferably, the Ig-like domain of the present invention contains two disulfide bonds between Cis-2-Cis-77 and Cis-22-Cis-28 of SEQ ID NO: 4, preferably between Cis-2-Cis-28 and Cis -22-Cis-77 of SEQ ID NO: 4.

The Ig-like domain of the membrane bound CSF3R is also described as expressed in SEQ ID NO: 10. Preferably, the Ig type domain consists of amino acid residues 25 to 120 of SEQ ID NO: 10 (as expressed in SEQ ID NO: 12) or amino acid residues 25 to 117 of SEQ ID NO: 10.

Preferably, the polypeptides are soluble, that is, they do not contain a functional membrane binding sequence and therefore can circulate in body fluids. In a more preferred aspect, the polypeptides of the present invention are soluble polypeptides of natural origin.

Another object of the present invention thus lies in a variant of the isolated soluble CSF3R. Preferably, the isolated soluble variant of CSF3R is a naturally occurring soluble variant.

Also, preferred variants of the CSF3R polypeptide of the present invention retain the ability to bind the natural ligands of the CSF3R. Such polypeptides therefore function as agonists and / or antagonists and can be used to stimulate and / or inhibit CSF3R mediated activities, for example, in pathological conditions. Preferably, the polypeptides of the present invention function as CSF3 agonists.

Another object of the present invention lies in a polypeptide variant of the CSF3R isolated in accordance with the present invention, wherein the variant is selected from its mature form, its glycosylated form, its pegylated form, its functional equivalent or its characteristic fragment.

It should be understood that the term CSF3R also includes the functional equivalents of the above sequence, that is, naturally occurring polymorphisms, sequences originating from other species, as well as sequences comprising one or more amino acid modifications that do not affect substantial the protein function of CSF3R. Functional equivalents generally show an 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 2.

The sequence derived from the CSF3R ligand binding domain can be derived or comprise the sequence of all or a ligand binding portion of the CSF3R extracellular domain or its functional equivalent. As mentioned above, the functional equivalent designates any modified sequence comprising one or more amino acid deletions, additions and / or substitutions, preferably 1, 2, 3, 5, 10, 15, 20, 30, 50 or more deletions, additions and / or substitutions that retain the ability to bind to a CSF3R ligand.

A specific embodiment of the present invention is a CSF3R polypeptide variant that has or comprises SEQ ID NO: 4 or its functional equivalent that retains the agonist activity of CSF3R.

A specific embodiment of the present invention is a CSF3R polypeptide variant that has or comprises SEQ ID NO: 4 or its functional equivalent that retains the antagonistic activity of CSF3R.

The present application further discloses any polypeptide comprising a characteristic fragment of a variant of CSF3R polypeptide as disclosed above. A characteristic fragment designates a fragment of at least 5 consecutive amino acids comprising a binding sequence formed as a result of the intervening amino acid residues removed. Such characteristic fragment may preferably comprise up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or more consecutive amino acid residues of the variant, preferably containing the binding sequence defined above. Preferably, a fragment consists of at least 6, 7, 8, 10, 20, 30, 40, 60, 80, 100, 120, 140, 160, 180, 185, 187 or 188 amino acids of SEQ ID NO: 2. Preferably, the fragment consists of consecutive amino acids.

As used herein, the term "fragment" refers to a polypeptide having an amino acid sequence that is the same, but not all, part of the amino acid sequence of the CSF3R polypeptide or one of its functional equivalents. The fragments should comprise at least n consecutive amino acids of the sequence and depending on the particular sequence, n is preferably 7 or more, preferably 8, 10, 20, 30, 40, 60, 80, 100, 120, 140, 160, 180, 185, 187 or 188 amino acids of SEQ ID NO: 2. Small fragments can form an antigenic determinant.

Preferably, the minimum length of the fragment is 6, 10, 20, 30, 40, 50, 60, 68, 100, 150, 200, 250, 300, 350, 400, 450 or 500 amino acids.

Preferably, the maximum length of the fragment is 188, 187, 186, 185, 180, 170, 166, 165, 164, 163, 160, 155, 150, 125, 100, 75 or 50 amino acids.

Preferably, the minimum length of the fragment is 18, 30, 60, 90, 120, 150, 180, 204, 300, 450, 600, 750, 1050, 1200, 1350 or 1500 nucleic acids.

Preferably, the maximum length of the fragment is 564, 561, 558, 555, 540, 510, 498, 495, 492, 489, 480, 465, 450, 375, 300, 225 or 150 nucleic acids.

Preferably, the maximum length of a polypeptide is 189, 190, 192, 195, 200, 210, 230, 250, 300, 400, 500, 600, 650, 700, 800, 900, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 amino acids.

Preferably, the maximum length of a nucleic acid n is 567, 570, 576, 585, 600, 630, 690, 750, 900, 1200, 1500, 1800, 1950, 2100, 2400, 2700, 3000, 5000, 7500, 10,000, 20,000, 30,000, 50,000, 75,000 or 100,000 nucleic acids.

Preferably, the functional equivalent has at least 75% identity, preferably at least 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 4.

Preferably, the functional equivalent has an antigenic determinant in common with SEQ ID NO: 4 consisting of 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 26, 30, 34, 38 or more amino acids of SEQ ID NO: 4.

An "antigenic determinant" may be a part of a polypeptide of the present invention that binds to an antibody combination site or a T-cell receptor (TCR). Alternatively, an "antigenic determinant" may be a surface site of a polypeptide of the present invention to which a single antibody molecule binds. Generally an antigen has several or many different antigenic determinants and reacts with antibodies of many different specificities. Preferably, the antibody is immunospecific with respect to a polypeptide of the present invention. Preferably, the antibody is immunospecific with respect to a polypeptide of the invention that is not part of a fusion protein. Preferably, the antibody is immunospecific with respect to CSF3R or a fragment thereof. Antigenic determinants usually consist of surface groups of chemically active molecules, such as amino acids or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific loading characteristics. Preferably, the "antigenic determinant" refers to a particular chemical group in a polypeptide of the present invention that is antigenic, that is, that elicits specific immune response.

Preferably, the functional equivalent contains one or more amino acid modifications, one or more non-conservative substitutions, one or more conservative substitutions and / or one or more amino acid derivatives.

As used herein, "functional equivalent" refers to a protein or nucleic acid molecule that possesses functional or structural characteristics that are substantially similar to a polypeptide or nucleic acid molecule of the present invention. A functional equivalent of a protein may contain modifications depending on the need for such modifications to perform a specific function. The term "functional equivalent" is intended to include fragments, mutants, hybrids, variants, analogs or chemical derivatives of a molecule.

The functional derivative comprises at least a portion linked to one or more functional groups, which occurs as one or more side chains in the amino acid residues. Preferably, the portion is a portion of polyethylene (PEG). PEGylation can be carried out by known methods, such as those described in, for example, WO99 / 55377.

The polypeptides may contain amino acids other than the 20 amino acids encoded by genes, modified either by natural processes, by post-transcriptional processing or by chemical modification techniques that are well known in the art. Among the known modifications that may commonly occur in the polypeptides of the present invention are glycosylation, lipid binding, sulphation, carboxylation range, for example, of glutamic amino acid residues, hydroxylation and ADPribosylation. Other possible modifications include acetylation, acylation, amidation, covalent bonding of flavin, covalent bonding of a portion of hematine, covalent bonding of a nucleotide or nucleotide derivative, covalent bonding of a lipid derivative, bonding phosphatidylinositol covalent, crosslinking, cyclization, disulfide bond formation, demethylation, covalent cross bond formation, cysteine formation, pyroglutamate formation, formylation, GPI anchor formation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, preylation, racemization, selenoylation, the addition of amino acids to proteins mediated by transfer RNA such as arginylation and ubiquitination.

Modifications can occur anywhere in a polypeptide, including the peptide's main structure, amino acid side chains and amino or carboxyl ends. In fact, blocking the amino ends

or carboxyl in a polypeptide, or both, by covalent modification is common in naturally occurring and synthetic polypeptides and such modifications can be found in polypeptides of the present invention.

Modifications that occur in a polypeptide will often depend on how the polypeptide is made. For polypeptides that are performed recombinantly, the nature and extent of the modifications will be largely determined by the post-transcriptional modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence. of the polypeptide in question. For example, glycosylation patterns vary between different types of host cells.

The polypeptides of the present invention can be prepared in any suitable way. Such polypeptides include isolated naturally occurring polypeptides (eg, purified from cell culture), recombinantly produced polypeptides (including fusion proteins), synthetically produced polypeptides or polypeptides that are produced by combining these methods.

Functional equivalents include natural biological variants (e.g. allelic variants or geographic variants within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of CSF3R polypeptides. . Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or unconserved amino acid residue (preferably a conserved amino acid residue) and said substituted amino acid residue may or may not be encoded with the genetic code. . Such typical substitutions are found between Ala, Val, Leu and Ile; between Ser and Tr; between the acidic residues Asp and Glu; between Asn and Gln; between basic residues Lis and Arg; or between the aromatic residues Fe and Tir. Particularly preferred are variants in which several, that is, between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or only 1 amino acid are substituted, removed or added in any combination. Especially preferred are silent substitutions, additions or deletions that do not alter the properties and activities of the protein. Also, conservative substitutions are especially preferred in this regard.

Such mutants further include polypeptides in which one or more of the amino acid residues include a substituent group.

In accordance with the present invention, any substitution should preferably be a "conservative" or "safe" substitution, which is commonly defined as a substitution that introduces amino acids having quite similar chemical properties (eg, a positively charged basic amino acid must be replaced by another positively charged basic amino acid), to preserve the structure and biological function of the molecule.

There are many models in the literature in which the selection of conservative amino acid substitutions can be made based on statistical and physical-chemical studies on the sequence and / or structure of proteins (Rogov S I and Nekrasov A N, 2001). Protein design experiments indicate that the use of specific subgroups of amino acids can produce active and foldable proteins, which helps to classify "synonymous" amino acid substitutions, which can be more easily accommodated in protein structure and can be used. to detect functional and structural counterparts and paralogs (Murphy LR et al., 2000). Synonyms amino acid groups and preferred synonyms amino acid groups are shown in Table 1.

Specific non-conservative mutations can also be introduced into the polypeptides of the invention for different purposes. Mutations that reduce the affinity of the FN3 domain that contains proteins can increase their ability to reuse or recycle, which potentially increases their therapeutic strength (Robinson C R, 2002). The immunogenic epitopes opportunely present in the polypeptides of the invention can be exploited to develop vaccines (Stevanovic S, 2002), or they can be eliminated through the modification of their sequence by following known methods for the selection of mutations to increase protein stability and correct them (van den Burg B and Eijsink V, 2002; WO 02/05146, WO 00/34317 and WO 98/52976).

In a preferred alternative, the synonymous groups for the amino acid derivatives included in the mimetic peptides are those defined in Table 2. A non-exhaustive list of amino acid derivatives also includes aminoisobutyric acid (Aib), hydroxyproline (Hyp), 1,2,3,4-tetrahydro-isoquinoline-3-COOH, indoline2-carboxylic acid, 4-difluoro-proline, L-thiazolidine-4-carboxylic acid, L-homoproline, 3,4-dehydro-proline, 3, 4-dihydroxyphenylalanine, cyclohexyl glycine and phenylglycine.

The term "amino acid derivative" refers to an amino acid or a chemical entity similar to the amino acid other than the 20 genetically encoded amino acids of natural origin. In particular, the amino acid derivative may contain cyclic, branched, linear, substituted or unsubstituted alkyl portions and may include one or more heteroatoms. Amino acid derivatives can be produced again or obtained by commercial sources (Calbiochem-Novabiochem AG, Switzerland; Bachem, USA).

Several methodologies for incorporating unnatural amino acid derivatives into proteins are disclosed in the literature, using transcription systems both in vitro and in vivo, to probe and / or improve protein structure and function (Dougherty D A, 2000). The techniques for the synthesis and development of mimetic peptides, as well as non-peptide mimetics, are also well known in the art (Golebiowski A et al., 2001; Hruby VJ and Balse PM, 2000; Sawyer TK, in "Structure Based Drug Design ”, published by Veerapandian P, Marcel Dekker Inc., pg. 557-663, 1997).

"Identity" indicates that at any particular position in the aligned sequences, the amino acid residue is identical between sequences.

Generally, an identity greater than 30% between two polypeptides is considered an indication of functional equivalence. Preferably, the functionally equivalent polypeptides of the first aspect of the invention have a degree of sequence identity with CSF3R polypeptides greater than 85%, 90%, 95%, 98%, 98.5%, 99% or 99.5% respectively.

Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that shows any of the functional activities of the polypeptides of the present invention.

Preferably, the "functional equivalent" can be a protein or nucleic acid molecule that shows substantially similar activity compared to CSF3R or its fragments in a suitable assay to measure biological activity or function. Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that shows identical or greater activity compared to CSF3R or its fragments in an assay suitable for measuring biological activity or function. Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that shows an activity of 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or greater in comparison with the CSF3R or its fragments in a test suitable for measuring biological activity or function.

Preferably, the "functional equivalent" may be a protein or polypeptide capable of inhibiting an activity in vivo or in vitro substantially similar to the polypeptides of the present invention. Preferably, the "functional equivalent" may be a protein or a polypeptide capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the manner in which the corresponding portion of the polypeptides of the invention would. For example, a "functional equivalent" could, in an immunoassay, decrease the binding of an antibody to the corresponding peptide (ie, the peptide, the amino acid sequence that was modified to reach the "functional equivalent" of the polypeptide of the invention, or to the polypeptide of the invention itself, where the antibody was raised against the corresponding peptide of the polypeptide of the invention. The equimolar concentration of the functional equivalent will decrease the aforementioned bond of the corresponding peptide by at least about 5%, preferably between about 5% and 10%, more preferably between about 10% and 25%, even more preferably between about 25% and 50% and more preferably between about 40% and 50%.

For example, functional equivalents may be fully functional or may lack function in one or more activities. Therefore, in the present invention, variations may affect the function, for example, of the activities of the polypeptide that reflect its possession of an Ig-like domain.

The activity of a polypeptide of the present invention can be confirmed in at least one of the following assays:

in the modulation of the proliferation, survival and maturation of cells compromised with the neutrophil granulocyte lineage, or

in the modulation of the activation of multiple signal transduction pathways, which include signal transducers and transcription activators (STAT), Ras / Raf / Erk, phosphatidylinositol 3-kinase (PI3-K) / Akt, and also the Stat proteases such as Stat5 protease, or

in the improvement of cardiac function, or

in the modulation of the induction of anti-apoptotic proteins, the inhibition of apoptotic cardiomyocyte death after myocardial infarction and the improvement of cardiac function, or

in reducing apoptosis of endothelial cells and increasing vascularization in the heart affected by infarction as protection against ischemic injury, or

in the survival of cardiac myocytes and in the prevention of remodeling of the left ventricle after myocardial infarction, or

in prolonged survival and increased activation of neutrophils combined with a sustained release of anti-inflammatory cytokines in patients suffering from community-acquired pneumonia (CAP), or

in synergy with the steel factor (ligand SF, Kit) or ligand Flt3 / Flk2 (FL) that helps the proliferation of primitive hematopoietic progenitors, or

in the inhibition of the biological activity of CSF3 in the formation of the normal bone marrow colony, in the inhibition of the stimulatory effect of CSF3 in the proliferation of leukemic progenitor cells to form colonies of leukemic blasts.

The administration of a polypeptide of the present invention to a patient to exercise one or more of the biological activities described above is also disclosed.

Alternatively, the activity of a polypeptide of the present invention can be confirmed in at least one of the following assays (as analyzed by Rutella et al. J. Immunol. 2005 Dec. 1; 175 (11): 7085-91):

in the modulation of proliferation and differentiation of normal stem cell hematopoietic cells (HSCs), or

in modulating neutrophil reconstitution after radiation-induced myelosuppression or chemotherapy, or

in the modulation of effector functions in mature neutrophils and the mobilization of bone marrow HSCs in the peripheral blood, or

in the interaction with the immune system by altering the reactivity of T cells and modifying the function of APC (for example, modulation of CSF that binds to human CD3 + T cells activated by mitogens and lines of lymphoblastoid T cells; for example, the modulation of the immunomodulatory genes of T cells, for example, GATA-3 and Stat5 that directly inhibit the production of proinflammatory cytokine by T cells at pharmacological doses), or

in the modulation of cytokine production (for example, the modification of cytokine production ex vivo by human white blood cells; for example, the modulation of the production of IL-1β, IL-12, IFN-γ, IL-18 , and TNF-α by whole blood and / or LPS-stimulated monocytes; for example, modulation of the LPS-induced release of the IL-1 receptor antagonist (IL-1ra) and soluble TNFR p55 and p75; for example, the Modulation of plasma levels of soluble TNFR and IL-1β compared to control individuals not previously treated with a polypeptide of the present invention in healthy volunteers who were subsequently given the endotoxin Salmonella abortus equi, which suggested effects anti-inflammatory in vivo; for example, modulation of TNF-α production by normal PBMC, activated by alloantigens at a posttranscriptional level; modulation of elevated hepatocyte growth factor s éricos, an angiogenic cytokine that can contribute to the formation of microvessels induced by CSF3; for example, modulation of the levels of soluble human HLA Ags, for example, HLA-G and HLA class I, which is said to modulate the alloimmune responses), or

in the in vitro differentiation of dendritic cells (DC) with tolerogenic capacity, or

in the modulation of T cell functions (for example, in healthy individuals, modulation of inhibition of T cell proliferation in response to mitogens by releasing soluble inhibitory factors; for example, capacity modulation of NKT cells to expand the in vitro response to αgalactosylceramide; for example, the mobilization of regulatory bone T cells (Treg) CD4 + CD25 + FoxP3 + from the functional bone marrow, as well as the natural T cell suppressor CD4 - CD8 - TCRα + β +, modulation of the expression of CXCL12 / SDF-1α, a CXCR4 ligand, in the bone marrow, thereby modulating the traffic of CD4 + CD25 + Treg cells; for example, the modulation of the mitogenic responses of T cells and cell-mediated cytotoxicity of the lymphokine-activated destroyer, which has been correlated with the expression of individual HLA alleles), or

in the induction of Treg cells (for example, the modulation of the generation of human Tr1 cells; for example, the modulation of the generation of Treg cells that produce IL-10 and the promotion of transplant tolerance), or

in the modulation of the number and function of DC (for example, the mobilization of DC2, which induces non-activated allogeneic T cells to produce IL-4 and IL-10; for example, the modulation of CCR7 expression in the blood dendritic cells, which affects the ability to migrate and return to their place of origin of DC; for example, modulation of IL-12 production by recovering DC after myeloablative HSC transplantation; for example, the modulation of the production of IL-10 and IFN-α, which are involved in the differentiation of Treg cells, for example, in the path of differentiation of human DCs derived from monocytes), or

in the expansion of myeloid precursors such as tolerogenic APCs (for example, in the expansion of a murine GM precursor population with regulatory activity; for example, in the modulation of the development of GVHD when it is cotransplanted in allogeneic recipient animals; for example, in in vitro differentiation of Treg cells specific for host Ags favored by the expansion of GM cells), or

in the modulation of HSC transplantation (for example, through the use of a hybrid resistance system in which NK cells mediate vigorous rejection, modulation of the insertion of parental bone marrow cells and the appearance of splenic colonies of the donor by continuous pretreatment of hybrid mice F 1; for example, the acceptance of rat bone marrow cells xenotransplanted in transgenic mice expressing a polypeptide of the invention; for example, in the down-regulation of proliferation of T cells to mitogens and alloantigens and decreased production of type 1 cytokine by T cells in mice; for example, the release of IL-4 by T cells treated with a polypeptide of the invention, which is consistent with a change in the pattern of cytokine Th2; for example, the ability of donor cells to mediate acute GVHD and improvement in total survival in ratone s receptors after pretreatment of donor mice with a polypeptide of the invention; for example, the increase in the frequency of CD4-CD8-NK1.1 + T cells responsible for the secretion of large amounts of IL-4 in donor mice treated with a polypeptide of the invention), or

in the stimulation of the neuronal differentiation of parents, neurogenesis and improvement of the result in the behavior after an ischemic lesion.

The administration of a polypeptide of the present invention to a patient to exercise one or more of the biological activities described above is further described.

To confirm the biological activity of a polypeptide of the present invention, various known assays can be used separately or in combination. In Schneider et al. J Clin Invest. August 2005; 115 (8): 2083-98, in Schneider et al. Cell Cycle December 2005; 4 (12): 1753-7 and an example for determining the function of a polypeptide of the present invention is described in US2005 / 0142102. For example, methods to confirm the function of sCFS3R include a colony formation assay that uses murine bone marrow cells; stimulation of bone marrow cell proliferation induced by sCFS3R; specific bioassays with cell lines that can depend on sCFS3R for growth or that can respond to sCFS3R (for example, AML-193; 32D; BaF3; GNFS-60; HL-60, MI; NFS-60; OCI / AML1a; and WEHI-3B). These and other assays are described in Braman et al. Am. J. Hematology 39: 194-201 (1992); Clogston C L et al Anal Biochem 202: 375-83 (1992); Hattori K et al Blood 75: 1228-33 (1990); Kuwabara T et al Journal of Pharmacobiodyn 15: 121-9 (1992); Motojima H et al Journal of Immunological Methods 118: 187-92 (1989); Sallerfors B and Olofsson European Journal of Haematology 49: 199-207 (1992); Shorter S C et al Immunology 75: 468-74 (1992); Tanaka H and Kaneko Journal of Pharmacobiodyn. 15: 359-66 (1992); Tie F et al Journal of Immunological Methods 149: 115-20 (1992); Watanabe M et al Anal. Biochem 195: 38-44 (1991).

In a more preferred aspect, the activity of a polypeptide of the present invention is confirmed by measuring the proliferation of primitive hematopoietic progenitors, by determining the number of colonies formed per plate in the course of one, two or three days in the presence of a polypeptide variant of the CSF3R isolated from the present invention (see Ku et al.). Preferably, the activity is confirmed if at least 1, 2, 3, 5, 7, 10, 15, 20, 25, 30 or more colonies are formed per plate. Preferably, colonies form in one day. Preferably, any active amount of CSF3R polypeptide can be used.

The present invention also relates to fusion proteins that comprise a CSF3R polypeptide as disclosed above, operably linked to an additional amino acid domain. The additional amino acid domain can be located upstream (N-ter) or downstream (C-ter) of the CSF3R polypeptide sequence. The additional domain may comprise any functional region, which provides, for example, an increase in the stability, focus or bioavailability of the fusion protein; which facilitates the purification or production, or confers on the additional biological activity of the molecule. Typical examples of such additional amino acid domains include, by way of non-taxation, a marker, a targeted peptide, a constant region of an immunoglobulin, a multidimerization domain and / or a biologically active protein or a fragment of this or a protein hormone heterodimeric such as human chorionic gonadotropin (hCG) as described in U.S. Patent No. 6,193,972. The term "operably linked" indicates that the polypeptide and the additional domain of amino acids are associated through a peptide bond, either directly or by spacer residues (eg, one or more Gly-Ser motifs). In this way, the fusion protein can be produced recombinantly, by direct expression in a host cell of a nucleic acid molecule that encodes it, as will be described below. Also, if necessary, the additional amino acid sequence included in the fusion proteins can be removed, either at the end of the production / purification process or in vivo, for example, by a suitable exopeptidase. For example, a spacer sequence included in the fusion protein may comprise a recognition site for an endopeptidase (such as a caspase) that can be used to separate the desired polypeptide variant from the additional amino acid domain by enzymatic cleavage, either in vivo or in vitro

Specific examples of additional amino acid residues include a marker sequence selected, for example, from a GST sequence and a His marker sequence. The marker sequence may be linked to the C-terminus or the N-terminus of the CSF3R polypeptide variant, preferably to the C-terminus.

In a particular embodiment, additional amino acid residues function as a signal peptide that directs protein secretion. The CSF3R is a type I transmembrane protein. The polypeptide variants of the CSF3R of the present invention can, however, be fused with a heterologous signal sequence, at the N-terminus of the polypeptide, to allow or increase secretion thereof. Said signal peptide may be any functional sequence in a host cell selected as a eukaryotic host cell (eg, mammalian) or prokaryotic. Examples of such signal peptides are known in the art.

In another particular embodiment, the additional amino acid residues in the fusion protein comprise an amino acid sequence derived from the constant region of an immunoglobulin, particularly the Fc portion of a human immunoglobulin. The sequence of the Fc portion can be derived, for example, from an IgG, preferably from a human IgG. Preferably, it is fused with heavy chain regions, such as the CH2 and CH3 domains, optionally with the human IgG1 axis region, for example. The Fc part may, for example, be mutated to prevent unwanted activities, such as complementary binding, binding to Fc receptors, or the like. Therefore, said Ig sequence can also be modified to reduce the effector function or to increase the stability of a resulting dimer. The amino acid sequence derived from the constant region of an immunoglobulin may be linked to the C-terminus or the N-terminus of the CSF3R polypeptide variant, preferably the C-terminus.

The generation of specific fusion proteins comprising the polypeptide of the first aspect of the invention and a portion of an immunoglobulin are described in example 11 of WO 99/09063, for example. Other isoforms of Ig molecules are also suitable for the generation of fusion proteins in accordance with the present invention, such as IgG 2 or IgG 4 isoforms, or other classes of Ig, such as IgM or IgA, for example. Fusion proteins can be monomeric, multimeric, hetero, or homomultimeric.

Other fusion proteins of the polypeptide of the first aspect of the invention can be prepared by the fusion of domains isolated from other proteins allowing the formation of dimers, trimers, etc. Examples of protein sequences that allow multidimerization of the polypeptides of the invention are isolated domains of proteins such as hCG (WO 97/30161), collagen X (WO 04/33486), C4BP (WO 04/20639), Erb proteins (WO 98/02540), or super spiral peptides (WO 01/00814). The polypeptides may contain amino acids other than the 20 amino acids encoded by genes, modified either by natural processes, by post-transcriptional processing or by chemical modification techniques known in the art. Among the known modifications that may commonly occur in the polypeptides of the present invention are glycosylation, lipid binding, sulphation, carboxylation range, for example, of glutamic amino acid residues, hydroxylation and ADP-ribosylation. Other potential modifications include acetylation, acylation, amidation, covalent bonding of flavin, covalent bonding of a portion of hematine, covalent bonding of a nucleotide or nucleotide derivative, covalent bonding of a lipid derivative, bonding phosphatidylinositol covalent, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cysteine formation, pyroglutamate formation, formylation, GPI anchor formation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, preylation, racemization, selenoylation, the addition of amino acids to proteins mediated by transfer RNA such as arginylation and ubiquitination.

In another particular embodiment, the additional amino acid residues in the fusion protein comprise a multidimerization domain, which allows the complexes to form between two or more fusion proteins of the present invention or between one or more fusion proteins of the present invention and a different protein. An example of such multidimerization domains includes a leucine zipper. The multidimerization domain may be linked to the C-terminus or the N-terminus of the CSF3R polypeptide variant, preferably to the C-terminus.

It should be understood that the fusion proteins of the present invention may comprise only one of the above additional amino acid residues or a combination thereof. For example, a fusion protein may comprise a signal peptide and a marker sequence, or a signal peptide and a multidimerization domain or a signal peptide and a constant region of an immunoglobulin, or a marker and the constant region of an immunoglobulin. Also, as indicated above, some of the additional amino acid sequences may be linked to the polypeptide variant of the CSF3R by spacer residues, particularly by cleavable spacer residues which allows subsequent separation of these elements if necessary. Such fusion proteins can be produced by any conventional technique known in the art, as will be discussed below.

Therefore, in accordance with an embodiment of the present invention, a hybrid fusion protein is provided which is a multimeric protein comprising more than one fusion polypeptide. Therefore, the fusion proteins described above can form homodimers and heterodimers and these are also included in the invention. The hybrid fusion protein may contain two, three, four or more fusion polypeptides in accordance with the first embodiment of the sixth aspect of the invention described above. Preferably, the hybrid fusion protein is a dimer comprising two fusion polypeptides as described above. If the fusion protein is a dimer, it can be a homodimer or a heterodimer. Preferably, the hybrid fusion protein comprises or consists of more than one fusion polypeptide selected from the above fusion polypeptides. The invention therefore includes homodimers comprising two fusion polypeptides. The invention further includes heterodimers comprising combinations of these sequences.

Non-immunoglobulin-derived proteins such as heterodimeric proteinaceous hormones can therefore be used to prepare fusion proteins. Fusion proteins employ α and β chains of a heterodimeric hormone or a portion thereof as scaffolding to which a polypeptide of the present invention is bound. An example of a heterodimeric proteinaceous hormone is the human chorionic gonadotropin hormone (hCG), which is a stable secreted protein with a long half-life. Examples of hybrid proteins that employ hCG can be found in U.S. Patent No. 6,194,177 and Campbell et al.

In general, the hybrid proteins of the invention include at least two polypeptide chains, wherein each polypeptide chain includes at least one polypeptide of the present invention linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof. Examples of heterodimeric proteinaceous hormones for use in the present invention include non-taxatively FSH, inhibin, TSH, hCG and LH.

In some embodiments, one of the subunits of the heterodimeric proteinaceous hormone in the hybrid protein comprises one or more alterations that reduce or eliminate the biological activity of the hormone, while preserving the ability of the altered subunit to dimerize with another subunit of the hormone. . In some embodiments, an altered subunit is an alpha subunit of hCG that comprises the removal of amino acids 88-92 (from 88-92), designated des88-92, which produces the biologically inactive hCG; nevertheless, it preserves the ability of the alpha subunit to dimerize with the beta subunit of hCG (the removal of only five residues at the carboxyl-terminal end of a subunit of hCG can effectively eliminate its biological activity while preserving its ability of forming heterodimers). In another embodiment, an altered subunit is an alpha subunit that comprises replacing a cysteine residue at an amino acid position 26 with an alanite (C26A). In another embodiment, an altered subunit is an alpha subunit comprising the removal of amino acids 88-92 and the substitution of a cysteine residue at an amino acid position 26 with an alanite (C26A). In another embodiment, an altered subunit is a beta subunit comprising a removal of amino acids 104-145 (from 104-145). The hybrid proteins of the invention may comprise: a) an altered alpha subunit and an unaltered beta subunit; b) an altered alpha subunit and an altered beta subunit; c) an unaltered alpha subunit and an altered beta subunit; or d) an unaltered alpha subunit and an unaltered beta subunit.

The fusion protein polypeptides of the invention can be found in isolation or in the form of active conjugates or their complexes.

In this regard, a particular object of the present invention lies in a conjugate comprising a variant of CSF3R polypeptide or a fusion protein as defined above. The conjugate comprises at least one chemical group (covalent) coupled to a polypeptide, such as a label, stabilizer, toxin, drug, etc. In a particular embodiment, the conjugate comprises a molecule selected from radioactive tags, biotin, fluorescent tags, cytotoxic agents, drugs or drug delivery agents, covalently coupled to any amino acid residue of the CSF3R polypeptide variant. Useful conjugates can be generated by the use of molecules and methods known per se in the art, for example, to allow the detection of interaction with a ligand (fluorescent or radioactive tags, biotin), or to improve agents in terms of efficacy of drug administration, such as polyethylene glycol and other natural or synthetic polymers (Harris JM and Chess RB, 2003; Greenwald RB et al., 2003; Pillai 0 and Panchagnula R, 2001).

The present application describes a receptor complex comprising a variant of CSF3R polypeptide or a fusion protein or conjugate as defined above. Such receptor complexes generally comprise a multimer formed between two or more fusion proteins of the present invention, or between one or more fusion proteins of the present invention and a different protein. Multimerization can be obtained by particular multidimerization domain (s) contained in the proteins, as described above. Such multimers can be formed in vitro, or they can be formed in vivo, after administration to an organism.

The polypeptides and fusion proteins of the present invention can be produced by any technique known per se in the art, such as by recombinant technologies, chemical synthesis, cloning, ligation, or combinations thereof.

In a particular embodiment, the polypeptides or fusion proteins are produced by recombinant technologies, for example, by the expression of a corresponding nucleic acid in a suitable host cell.

In this regard, the term "nucleic acid molecule" comprises any nucleic acid molecule that encodes a polypeptide or a fusion protein as disclosed above. The nucleic acid can be a DNA (for example, cDNA, gDNA, synthetic DNA, etc.) an RNA (for example, mRNA), an APN (nucleic peptide acid), etc., more preferably a DNA, even more preferably a cDNA molecule. A particular object of the present invention lies more specifically in a nucleic acid molecule that comprises or consists of a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, or a complementary strand or its degenerate sequence.

The term "purified nucleic acid molecule" preferably refers to a nucleic acid molecule of the invention that (1) was separated from at least about 50 percent proteins, lipids, carbohydrates or other materials with which it naturally occurs. when all of the nucleic acid is isolated from the source cells, (2) it is not bound to all or a portion of a polynucleotide to which the "purified nucleic acid molecule" is naturally bound, (3) is operatively linked to a polynucleotide that is not naturally linked or (4) does not occur naturally as part of a larger sequence of polynucleotides. Preferably, the isolated nucleic acid molecule of the present invention is substantially free of any other contaminant nucleic acid molecule or other contaminants that are in its natural environment that may interfere with its use in the production of polypeptides or its therapeutic use, diagnostic, prophylactic or research. In a preferred embodiment, genomic DNA is specifically excluded from the scope of the present invention. Preferably, genomic DNA greater than 10 kbp (kilo base pairs), 50 kbp, 100 kbp, 150 kbp, 200 kbp, 250 kbp or 300 kbp is specifically excluded from the scope of the invention. Preferably, the "purified nucleic acid molecule" consists solely of cDNA.

A degenerate sequence designates any nucleotide sequence that encodes the same amino acid sequence as a reference nucleotide sequence, but comprises a different nucleotide sequence as a result of the degeneracy of the genetic code.

A further object of the present invention is a vector comprising a nucleic acid sequence as defined above. The vector can be any cloning or expression vector, which replicates integrally or autonomously, that is functional in any prokaryotic or eukaryotic cell. In particular, the vector can be a plasmid, cosmid, virus, phage, episome, artificial chromosome and the like. The vector may comprise regulatory elements, such as promoters, terminators, enhancers, selection markers, origin of the replica, etc. Specific examples of such vectors include prokaryotic plasmids, such as pBR, pUC or pcDNA plasmids; viral vectors, including retroviral, adenoviral or AAV vectors; bacteriophages, baculovirus, BAC or YAC, etc. as will be discussed below.

A further aspect of the present invention is a recombinant host cell, wherein said cell comprises a nucleic acid molecule or a vector as defined above. The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic cells include bacteria such as E. coli. Examples of eukaryotic cells are yeast cells, plant cells, mammalian cells, including any primary cell culture.

or established cell line (for example, 3T3, Vero, HEK293, TN5, etc.). Particularly preferred mammalian cells of the present invention are CHO cells.

A further object of the present invention is a method for producing a variant of CSF3R polypeptide or fusion protein as defined above, the method comprises culturing a recombinant host cell of the invention under conditions that allow the expression of the molecule of nucleic acid and the recovery of the polypeptide produced. The polypeptide can be recovered from the cell culture supernatant if the polypeptide is secreted, or from the cytoplasm or cellular debris, if appropriate. The polypeptide product can be glycosylated or not, or contain other post-translational modifications depending on the host cell used. Preferred glycosylation sites lie in amino acid 51, 93, 128, 134, 389, 474, 579, 610 of SEQ ID NO: 10.

Many books and reviews provide demonstrations of how to clone and produce recombinant proteins through the use of prokaryotic or eukaryotic host cells and vectors, such as some titles in the “A Practical Approach” series published by Oxford University Press (“DNA Cloning 2: Expression Systems ”, 1995;“ DNA Cloning 4: Mammalian Systems ”, 1996;“ Protein Expression ”, 1999;“ Protein Purification Techniques ”, 2001).

In general, the vectors can be episomal or non-homologous integration vectors, which can be introduced into the appropriate host cells by any suitable means (transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, etc. .) for its transformation. The factors of importance in the selection of a particular plasmid, viral or retroviral vector include: the ease with which the recipient cells containing the vector can be recognized and selected from those recipient cells that do not contain the vector; the number of copies of the vector that are desired in a particular host; and if it is desired that it is possible to "move" the vector between host cells of different species. These vectors should allow the expression of the fusion polypeptide or proteins of the invention in prokaryotic or eukaryotic host cells, under the control of suitable transcriptional start / end regulatory sequences, which are chosen to be constitutively active or inducible in said cell. A substantially enriched cell line in said cells can be isolated to provide stable cell lines.

A particularly preferred method of high yield production of a recombinant polypeptide of the present invention is performed by using the dihydrofolate reductase (DHFR) amplification in DHFR-deficient CHO cells, by using successively increased levels of methotrexate as described in U.S. Patent No. 4,889,803. The polypeptide obtained can be in glycosylated form.

For eukaryotic host cells (for example, yeast, insect or mammalian cells), different transcriptional and transnational regulatory sequences may be employed, depending on the nature of the host. They can be derived from viral sources, such as adenovirus, papillomavirus, ape virus or the like, where regulatory signals are associated with a particular gene that has a high level of expression. Examples are the TK promoter of the Hepes virus, the initial SV40 promoter, the yeast gene promoter gal4, etc. Transcriptional initiating regulatory signals that allow repression and activation can be selected so that gene expression can be modulated. Cells that were stably transformed by the introduced DNA can be selected by introducing one or more markers that allow the selection of host cells containing the expression vector. The label can provide phototrophy to an autotrophic host, resistance to biocides, for example, antibiotics or heavy metals such as copper or the like. The selectable marker gene can be directly linked to the DNA sequences that will be expressed (for example, in the same vector) or introduced into the same cell by co-transfection. Additional elements may also be necessary for optimal protein synthesis of the invention.

Particularly suitable prokaryotic cells include bacteria (such as Bacillus subtilis or E. coli) transformed with a recombinant bacteriophage, plasmid or cosmid DNA expression vector. Said cells generally produce proteins comprising an N-terminal methionine residue and said proteins represent particular objects of the present invention. Preferred cells for use in the present invention are eukaryotic host cells, for example, mammalian cells, such as human, monkey, mouse, Chinese Hamster Ovary (CHO) cells, because they provide post-translational modifications to protein molecules, including the correct folding or glycosylation at the correct sites. Alternative eukaryotic host cells are yeast cells (eg, Saccharomyces, Kluyveromyces, etc.) transformed with yeast expression vectors. Also, yeast cells can make post-translational peptide modifications that include glycosylation. There are several recombinant DNA strategies that use strong promoter sequences and a large copy number of plasmids that can be used for the production of the desired proteins in yeast. Yeast cells recognize the precursor sequences in cloned mammalian gene products and secrete polypeptides that have precursor sequences (i.e., pre-peptides).

For stable long-term production of a recombinant polypeptide stable expression is preferred. For example, cell lines that stably express the polypeptide of interest can be transformed by using expression vectors that may contain viral origins of replication and / or endogenous expression elements and a selectable marker gene in the same vector or in a vector. separate vector. After the introduction of the vector, the cells can be cultured for 1-2 days in an enriched medium before switching to the selective medium. The purpose of the selectable marker is to confer resistance to selection and its presence allows the growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells can be proliferated by using tissue culture techniques suitable for the cell type. A substantially enriched cell line in said cells can be isolated to provide stable cell lines.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available in the American Type Culture Collection (ATCC), which include non-taxatically, Chinese hamster ovary (CHO) cells, from HeLa, hamster breeding kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) and other cell lines. In the baculovirus system, materials for baculovirus / insect cell expression systems are commercially available as an Invitrogen kit, among others.

In addition to recombinant DNA technologies, the polypeptides or fusion proteins of the present invention can be prepared by chemical synthesis technologies. Examples of chemical synthesis technologies are solid phase synthesis and liquid phase synthesis. As a solid phase synthesis, for example, the amino acid corresponding to the carboxyl end of the polypeptide to be synthesized is bound to a support that is insoluble in inorganic solvents and by alternating repetition of reactions (for example, by sequential condensation of amino acids with their amino groups and side chain functional groups protected by suitable protecting groups), the polypeptide chain is extended. Solid phase synthesis methods are broadly classified by the tBoc method and the Fmoc method, depending on the type of protective group used. Fully synthetic proteins of comparable size to that of CSF3R are disclosed in the literature (Brown A et al., 1996).

The polypeptides of the present invention can be produced, formulated, administered or used generically in other alternative forms that may be preferred in accordance with the desired method of use and / or production. The proteins of the invention can be modified post-translationally, for example by glycosylation. The polypeptides or proteins of the invention can be provided in active isolated form (or purified), or as precursors, derivatives and / or salts thereof.

As indicated above, the term "active" or "biologically active" means that said polypeptides have the ability to bind a ligand of CSF3R and function as an agonist or antagonist of CSF3R. A "biologically active" polypeptide of the present invention is also one that shows activity in at least one of the aforementioned assays.

"Precursors" are compounds that can be converted into the polypeptides of the present invention by metabolic and / or enzymatic processing before or after administration to cells or to an organism.

The term "salts" herein refers to both salts of carboxyl groups and acid addition salts of amino groups of the polypeptides of the present invention. The salts of a carboxyl group can be formed through means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts and the like, and salts with organic bases such as those formed, for example , with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid and salts with organic acids such as acetic acid or oxalic acid. Any of said salts has activity substantially similar to the polypeptides of the invention.

The term "derivatives" as used herein refers to derivatives that can be prepared from functional groups present in the side chains of the amino acid portions or in the amino or carboxyl terminal groups in accordance with the methods known per se. in the technique Such derivatives include, for example, aliphatic esters or amides of the carboxyl groups and N-acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl groups and are formed with acyl groups such as, for example, alkanoyl or aroyl groups.

The purification of the fusion polypeptides or proteins of the invention can be carried out by a variety of methods known per se in the art, such as non-taxatively, any conventional procedure that involves extraction, precipitation, chromatography, electrophoresis or the like. . A particular purification procedure is affinity chromatography, through the use of antibodies (monoclonal) or affinity groups that selectively bind to the polypeptide and that are generally immobilized in a gel matrix contained within a column. Purified preparations of the proteins of the invention, as used herein, refer to preparations containing less than 15% contaminants, more preferably comprising at least 90, 95 or 97% of the polypeptide. An isolated protein, polypeptide or nucleic acid denotes a protein, polypeptide or nucleic acid that is not found in its natural environment.

The polypeptides of the invention are useful by themselves, as components of fusion proteins such as Fc fusion and / or in combination with another agent. Preferably, the Fc fusion comprises SEQ ID NO: 4.

Preferably the agent is selected from interferon-beta, CSF3, GM-CSF, tissue plasminogen activator, hematopoietic factor, soluble NgR members (for example Nogo-66), antagonists (for example, antibodies) directed to NgR, antagonists (for example , antibodies) directed to myelin inhibitors (eg, Nogo, MAG or Omgp), steel factor (SF ligand, Kit), ligand Flt3 / Flk2 (FL), RhoA blockers, Rho blockers, ROCK blockers, channel blockers sodium, growth-related proteins such as BDNF, VEGF, erythropoietin (EPO), neurotrophin-3 (NT-3) or GAP-43, β-secretase modulators, CXCL10, serotonin receptor agonists (for example, 5-5 -HT1λ / 2A / 7), LIF, EGFR blockers such as Erlotinib and / or methylprednisolone. Interferon-beta is particularly preferred.

Several combinations may be provided: 1) the combination of a polypeptide of the present invention with T cell activation inhibitors such as anti-CD3 Ab; 2) the combination of a polypeptide of the present invention with other hematopoietic factors, such as Flt3-L, either separately or in combination in a chimeric molecule (progenipoietin); 3) the classification of a particular cellular subgroup induced by a polypeptide of the present invention, which can provide protection against autoimmune and allooreactive responses; and 4) the classification of a particular cell subgroup mobilized by a combination of growth factors, including G-CSF and a polypeptide of the present invention, (for example, the particular subgroups of immature hematopoietic progenitor cells, mobilized by a combination of growth factors, which include the G-CSF and a polypeptide of the present invention, may have the ability to stop the onset of symptomatic diabetes).

A further object of the present invention is a pharmaceutical composition comprising a product (for example, a polypeptide, a fusion protein, a conjugate, a receptor complex, a nucleic acid, a cell or vector) as defined above and a vehicle. or pharmaceutically acceptable diluent. The most preferred pharmaceutical compositions of the present invention comprise a polypeptide comprising SEQ ID NO: 4

or a fusion protein comprising said polypeptide.

The use of a product (for example, a polypeptide, a fusion protein, a conjugate, a nucleic acid molecule, a vector or a cell) as disclosed above is disclosed for the manufacture of a pharmaceutical composition for treating a human subject

GCSF is generally used for the treatment of different types of neutropenia in humans. It is one of the few growth factors approved for clinical use. In particular, it is used to reduce chemotherapy-induced cytopenia (Viens et al., J. de Clin. Oncology, Vol. 20, No. 1, 2002: 24-36). GCSF was also used for therapeutic use of infectious diseases as a possible adjuvant agent (Hubel et al., J. de Infectious Diseases, Vol. 185: 1490-501, 2002).

CSF3 agonists used for therapy are known. For example, Amgen Inc launched pegfilgrastim, a pegylated form of recombinant CSF3, for the treatment of chemotherapy-induced neutropenia or leukopenia. Bolder Biotechnology developed a fusion protein comprising the pegylated CSF3 bound to IgG-Fc called BBT-001 for the treatment of leukopenia. Chugai Pharmaceutical launched lenograstim, a recombinant human CSF3 for the treatment of leukopenia, hematologic disease, immune disorder, neutropenia and viral infection. Dragon Pharmaceuticals developed a formulation of CSF3 for the treatment of leukopenia. Kancer limited developed an oral formulation of CSF3 for the treatment of leukopenia. Kyowa Hakko Kogyo Co Ltd launched nartograstim (injectable CFS3) for the treatment of anemia or leukopenia. Neose Technologies developed the long acting glycopegylated CSF3 for the treatment of neutropenia. Transkaryotic Therapies Inc developed the gene-activated CSF3 (GA-GCSF) for the treatment of neutropenia.

In a mouse model of myocardial infarction, treatment with CSF3 did not affect the magnitude of the initial infarction on day 3 but improved heart function 1 week after the infarction and the beneficial effects were less given the delay in the beginning of treatment ( Harada et al. Nature Med. Vol. 11: 305-311, 2005). CSF3 induced antiapoptotic proteins and inhibited apoptotic cardiomyocyte death. Likewise, CSF3 reduced apoptosis of endothelial cells and increased vascularization in hearts affected by heart attack. Harada et al. He suggested that CSF3 promotes the survival of cardiac myocytes and prevents remodeling of the left ventricle after myocardial infarction through functional communication between cardiomyocytes and non-cardiomyocytes.

The application of a unit dose of G-CSF in patients suffering from community-acquired pneumonia (CAP) caused by prolonged survival and increased activation of neutrophils combined with a sustained release of anti-inflammatory cytokines (Droemann et al. Respiration 2005 Dec. 12. Epub prior to publication).

CSF3 is also beneficial in animals for the prevention and / or treatment of immunological diseases, for example, graft versus host disease, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease and diabetes (Rutella et al. J. Immunol. 2005 Dec. 1; 175 (11): 7085-91).

In graft versus host disease, in vitro response of donor cells 5 days after G-CSF administration can predict the occurrence of acute GVHD in transplant patients and donor T cells with significant G-induced suppression. -CSF of in vitro allooreactivity induces less frequently the GVHD in the receptors. The production of TGF-β after allogeneic HSC transplantation can provide an additional explanation for the opposing effects of G-CSF administration to donors of acute versus chronic GVHD. In this regard, administration of G-CSF to donor mice may induce TGF-β-e IL-10-dependent protection against acute GVHD but contribute to TGF-β-dependent exacerbation of chronic GVHD. The neutralization of TGF-β in transplanted mice protects from chronic GVHD in the gastrointestinal tract and the skin but not from liver abnormalities.

In inflammatory bowel disease, G-CSF has been used successfully in experimental colitis in white rabbits in New Zealand. The animals were previously treated 24 hours before or during the induction of colitis with 50 or 200 μg / kg rG-CSF. The administration of cytokine at any dose resulted in increased levels of tissue myeloperoxidase, despite a histologically similar mucosal polymorphonuclear cell infiltrate in the G-CSF treated group compared to the colitis control group. Likewise, the dialysis fluid levels of leukotriene B4 and thromboxane B2 were significantly lower in the treated animals. Similar results were obtained in 2,4,6-trinitrobenzene sulfonic acid colitis in rats, a model of Th1 disease. The G-CSF at 250 μg / kg / day greatly attenuated both the loss of body weight and the thickening of the colonic wall due to progressive transmural inflammation. This effect was associated with a significant inhibition of the transcription of IFN-7 and IL-12p35. Preclinical data also transferred to the treatment of human inflammatory bowel disease. It was proved that G-CSF is effective in the acute endoscopic postoperative recurrence of Crohn's disease. Five patients were treated with 300 μg of human recombinant G-CSF three times per week for 12 weeks. G-CSF was found to be safe and was well tolerated and a significant increase in neutrophil counts, IL-1ra and TNFR p55 and p75 soluble in the plasma of patients treated with G-CSF was shown. In an open-label study, G-CSF demonstrated safety and efficacy for the treatment of active Crohn's disease. Patients received 300 μg of G-CSF for 12 consecutive weeks, after which a statistically significant decrease in disease activity was achieved. These studies suggest that rG-CSF may be a promising therapeutic approach to Crohn's disease.

The anti-inflammatory properties of G-CSF and its ability to change the cytokine profiles of T cells towards Th2 and to promote differentiation of DC and tolerogenic Treg cells influenced their therapeutic evaluation in experimental models of autoimmune diseases such as systemic lupus erythematosus ( SLE) and rheumatoid arthritis (RA). Nephritis, the final stage of lupus disease characterized by inflammation, was prevented in mice given a high-dose regimen of G-CSF (200 μg / kg), even when treatment was initiated in animals that They already suffered from proteinuria. A decoupling was observed between the deposition of the immune complex and kidney damage, which corresponded to a profoundly reduced expression of FcγRIII (CD16) within the glomeruli, receptors that mediate the inflammatory response of the mesangial cells of the kidney to Igs. Accordingly, the inflammatory response to serum IL-12 was limited and mortality was significantly delayed. In inflammatory arthritis, neutralization of endogenous G-CSF markedly reduced disease progression to the same extent as anti-TNF treatment. Cell therapy with hematopoietic progenitor cells mobilized by G-CSF, devoid of other pro-inflammatory cell subgroups induced by G-CSF may be therapeutically promising in patients with autoimmune diseases.

The beneficial effects of G-CSF in animal models of multiple sclerosis (MS) dependent on T-cells and type 1 autoimmune diabetes (T1D) proved to be of possible interest for translation in therapeutic strategies. The short 7-day treatment with G-CSF (200 μg / kg), initiated at the beginning of clinical signs, conferred lasting protection to SJL: J mice against experimental autoimmune encephalomyelitis induced by myelin basic protein. Protected mice showed limited demyelination and reduced recruitment of T cells in the central nervous system (CNS) as well as very discrete autoimmune inflammation and levels of cytokine and chemokine mRNA barely detectable in the CNS. These effects were based on immunoregulatory events that took place in the periphery and included an imbalance in the chemokine production rate (MIP-1α / MCP1, for example, CCL3 / CCL2) by macrophages and autoreactive lymphocytes that correlated with a deviation immune from the autoreactive response to Th2. A dramatic reduction in the production of systemic and lymphocytic TNF-α was also observed. The protective effect of G-CSF was confirmed in C57BL / 6 mice immunized with the MOG peptide. The high expression of G-CSF genes occurred selectively in brain lesions of patients in the acute phase of MS, which suggested an endogenous protective role of growth factor.

The present application discloses that a polypeptide of the present invention, alone and / or in combination with one or more additional factors can be used to treat multiple sclerosis (MS) and / or provide prophylactic neuroprotective therapy in patients suffering from multiple sclerosis. This is based on the presence of the GCSF receptor on oligodendrocytes, which supports the direct efficacy of GCSF in primary white MS cells. Likewise, the GCSF receptor is present in nerve cells and their processes, which are involved in later stages of the disease and may have a correlation with lasting disabilities (Cid. Et al. (2002), J Neurol Sci, 193, 103 -9). Even areas of the brain that appear normal with respect to changes in white matter show signs of degeneration. Therefore, axonal pathology and neurodegeneration are important therapeutic objectives in Multiple Sclerosis. GCSF with its anti-apoptotic activity in neurons and its pro-regenerative potential (by improving neurogenesis and plasticity) supports the fact that the compositions described herein can be used as new therapies to treat multiple sclerosis.

Likewise, the pathophysiological mechanisms in multiple sclerosis coincide with important mechanisms in cellular ischemia, for example, the participation of nitric oxide (Smith, et al. (2001), Ann Neurol, 49, 470-6) and the participation of glutamate excitoxicity (Pitt et al. (2000), Nat Med, 6, 67-70). In light of this information, the polypeptide disclosed herein represents a new treatment option for multiple sclerosis which, being not restricted by any particular mechanism or theoretical considerations, protects neurons directly as opposed to common treatments that reduce inflammation. .

The results that demonstrate the ability of G-CSF to counteract acute neuronal degeneration and to conduct neurogenesis in acute ischemia indicate that G-CSF is a promising drug for stroke, behavioral improvements and degenerative and autoimmune diseases of the system. nervous (Schneider et al. J Clin Invest. 2005 August; 115 (8): 2083-98; Schneider et al. Cell Cycle. 2005 December; 4 (12): 1753-7.US2005 / 0142102). The CSF3R exhibits a surprisingly broad neuronal expression pattern in the CNS. The G-CSF showed to be neuroprotective in 2 different models of focal cerebral ischemia with transient activation of STAT3 and ERK1 / 2 and a strong lasting activation of ERK5 linked to the promotion of neuronal survival as well as the activation of the PI3K / Akt pathway linked to the apoptotic activity of the G-CSF. The most surprising effect of G-CSF administered periferally in the brain was observed in the dentate gyrus, where G-CSF increased the number of newly generated neurons in ischemic conditions but also in animals of the non-ischemic reference surgical group. It is therefore fascinating to speculate that G-CSF can improve repair and structural function even in healthy individuals or at long intervals after stroke. The wide range of data from different laboratories underlines the very stable pro-regenerative and neuroprotective effect of G-CSF in several stroke models. G-CSF may also be suitable as a supportive treatment in the rehabilitation phases after stroke. A predominant role of G-CSF in the survival and differentiation of progenitor cells in the postischemic brain is also suggested. The cortical phototrombosis model used by Schneider et al. It exerts the most prominent impact on the sensorimotor behavior, which was also measured in the battery of tests performed, while the hippocampus formation is more frequently linked to the learning and memory processes. However, there is also a wealth of data that support a possible role of the hippocampus in the functional recovery of motor deficits. It was suggested that the increase in hypocampic neurogenesis induced by G-CSF directly affects the recovery of sensorimotor deficits induced by cortical lesions. Therefore, the signaling with G-CSF seems to be a new protective system in the brain that participates in the consideration of acute neurodegeneration and the regulation of the formation of new neurons.

Neurogenesis is a mechanism that can lead to increased plasticity of neural networks and can replace the gradual loss of neurons. Therefore, the improvement, increase or increase in the cognitive ability of an individual who suffers, shows and / or is believed to have some level of cognitive loss is described by administering one or more compositions as described herein. for the individual in accordance with the administration discussion herein. Cognitive improvement can also benefit those still useful individuals with non-pathological conditions, for example, individuals who do not have cognitive impairment.

The determination of cognitive ability and therefore its improvement is known to those skilled in the art. In case of measuring increases or improvements in cognitive ability, these are compared before administration of the compositions of the invention and after administration (and can also be measured during administration in some embodiments) by using the same test, for example, with the same criteria, parameters, etc.

Likewise, the use of the compositions disclosed herein for the improvement of cognition is not limited to the non-pathological deterioration of mental ability, but can also be applied to stimulate the normal physiological repertoire of mental abilities, for example, the improvement of memory, the improvement of fixed motor coordination and / or the improvement of logical capabilities.

The G-CSF also provided protection against experimental autoimmune diabetes in NOD mice. G-CSF (200 μg / kg) reversed the accelerating effects of cyclophosphamide, prevented the loss of CD4 + CD25 + Treg cells and nullified the robust cytokine, particularly IFN-γ and the chemokine blast shot into immune cells by cyclophosphamide. In the spontaneous diabetes model, the treatment of NOD mice at week 4 of age for 5 consecutive days that was repeated every 4 weeks thereafter until week 16 of age, the onset of the disease and destructive insulins were prevented in a lasting way. This protection corresponded to the marked recruitment of two large regulatory subgroups, that is, plasmocytoid dendritic cells and CD4 + CD25 + Treg cells. Also, G-CSF receptors showed an accumulation, particularly in the peripancreatic lymph nodes of CD4 + CD25 + Treg cells, which produced high levels of TGF-β1 and remained functional, which actively contained the transfer of diabetes into NOD-SCID secondary receptors. The classified dendritic cells of mice that were given a 5-day G-CSF treatment, relative to the dendritic cells of donors treated with excipients, were able, after adoptive transfer to secondary NOD receptors, to trigger an accumulation remarkably improved Treg CD4 + CD25 + cells that expressed significantly higher levels of membrane TGF-β1. Endogenous infection response factors such as G-CSF may be implicated in protection against the onset of T1D caused by infectious events, particularly if they are carried out in early stages of diabetes development.

Similarly, the activity of a polypeptide of the present invention can be demonstrated in the diseases mentioned above.

CD4 + CD25 + Treg cells mobilized by the G-CSF can represent a promising source of Treg cells for clinical application as they can expand up to 40,000 times with artificial APC and a high dose of IL-2. Similarly, a polypeptide of the present invention can mobilize CD4 + CD25 + Treg cells.

Therefore, CSF3 has been linked to the following diseases: graft versus host disease, cancer, neutropenia, neurological disorders or conditions, pneumonia, autoimmune diseases, hematological diseases, hematopoietic disorders, infectious diseases, inflammatory bowel disease and diabetes.

Neurological conditions can generally be classified into three classes: diseases with ischemic or hypoxic mechanisms referred to herein as diseases related to ischemia; neurodegenerative diseases (see Adams et al, Principles of Neurology, 1997, 6 th Ed., New York, pp 1048 ff); and neurological and psychiatric diseases associated with the death of neuronal cells. Other neurological conditions also include the improvement of cognitive ability and the treatment of brain tumors, such as glioblastomas, astrocytomas, meningiomas and neurinomas. Examples of neurological and psychiatric diseases associated with neuronal cell death include septic shock, intracerebral hemorrhage, subarachnoid hemorrhage, multiple infarction dementia, inflammatory diseases (such as vasculitis, multiple sclerosis and Guillain-Barre syndrome), neurotraumatism (such as spinal cord trauma and brain trauma), peripheral neuropathies, polyneuropathies, epilepsy, schizophrenia, depression, metabolic encephalopathies and central nervous system infections (viral, bacterial, fungal).

Diseases with ischemic or hypoxic mechanisms, referred to herein as diseases related to ischemia, can also be subclassified into general diseases and cerebral ischemia. Examples of such general diseases that involve ischemic or hypoxic mechanisms include myocardial infarction, heart failure, congestive heart failure, myocarditis, pericarditis, perimiocarditis, coronary heart disease (coronary artery stenosis), angina pectoris, congenital heart disease, shock, limb ischemia, ischemic injury, renal artery stenosis, diabetic retinopathy, thrombosis associated with malaria, artificial heart valves, anemias, hypersplenic syndrome, emphysema, pulmonary fibrosis and pulmonary edema. Examples of cerebral ischemic disease include strokes (such as hemorrhagic strokes), cerebral microangiopathy (microangiopathy), cerebral ischemia during childbirth, ischemia during / after cardiac arrest or resuscitation, cerebral ischemia due to intraoperative problems, cerebral ischemia during surgery of carotids, chronic cerebral ischemia due to stenosis of the arteries that provide blood to the brain, thrombosis of a venous sinus or thrombosis of the cerebral veins, malformations of the cerebral vessels and diabetic retinopathy.

Examples of neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, Wilson's disease, multiple system atrophy, Alzheimer's disease, Pick's disease, Lewy body disease, Hallervorden-Spatz's disease, torsion dystonia, hereditary sensorimotor neuropathies (HMSN), Gerstmann-Strussler-Schanker disease, Creutzfeld-Jakob disease, Machado-Joseph disease, Friedreich ataxia, non-Friedreich ataxias, Gilles de la Tourette syndrome, family tremors, olivopontocerebellar degenerations, paraneoplastic cerebral syndromes, hereditary spasmodic paraplegies, inherited optic neuropathy (Leber), retinitis pigmentosa, Stargardt's disease and Keams-Sayre syndrome. More preferably, the neurodegenerative disease is multiple sclerosis.

Preferably, graft versus host disease is acute GVHD or chronic GVHD. More preferably, graft versus host disease is acute GVHD. The CSF3 agonist is useful for acute GVHD and the CSF3 antagonist is useful for chronic GVHD.

Preferably, the inflammatory bowel disease is colitis or Crohn's disease (for example, acute endoscopic postoperative recurrence of Crohn's disease or active Crohn's disease).

Preferably, autoimmune disease is an autoimmune disease of the nervous system, systemic lupus erythematosus (SLE), osteoarthritis or rheumatoid arthritis (RA).

Preferably, diabetes is a type 1 autoimmune diabetes (T1D).

Preferably, pneumonia is community acquired pneumonia (CAP).

Preferably, neutropenia is a severe congenital neutropenia, chemotherapy-induced cytopenia (CT), leukopenia.

Preferably, cancer is a malignant disorder of myeloid lineage such as acute myeloid leukemia (AML), leukemia, leukemogenesis, myelodysplastic syndromes, myeloid leukemia, acute lymphocytic leukemia (ALL), acute leukemia, chronic myeloid leukemia (CML), solid tumors, epithelial ovarian carcinoma, neoplasms, bladder cancer, carcinoma, prostate cancer, squamous cell carcinoma, melanoma or colorectal cancer.

Preferably, the hematological disease is myocardial infarction.

Accordingly, a method for treating one or more of the aforementioned diseases in a mammal is also disclosed by administering to the mammal of a polypeptide of the present invention, its derivatives, its mimetics, its fusion proteins and its combinations or cells that They secrete these factors to treat the condition.

The above products and pharmaceutical composition are particularly suitable, for example, to treat graft versus host disease, cancer, neutropenia, neurological disorders or conditions, pneumonia, autoimmune diseases, hematological diseases, hematopoietic disorders, infectious diseases, inflammatory bowel disease and diabetes. .

A method for treating a neurological condition in a mammal is also described by preparing a composition of neuronal stem cells with a polypeptide of the present invention, its derivatives, its mimetics and combinations thereof and then administering the neuronal stem cells to a mammal for The treatment of the condition.

A method for treating a neurological condition in a mammal is also disclosed by administering to the mammal a polypeptide of the present invention to treat a neurological condition.

A method for improving the survival of a transplanted cell to a mammal is also disclosed by the introduction into a cell of one or more polynucleotides encoding a polypeptide of the present invention, its derivatives, its mimetics and / or combinations thereof before transplanting the cell to a mammal, whereby the cell expresses a polypeptide of the present invention in an amount sufficient to improve cell survival relative to cell survival prior to the introduction of polynucleotides.

A method for improving the viability of a neuronal cell culture is also described by providing a polypeptide of the present invention, its derivatives, its mimetics, its fusion proteins and / or combinations thereof to improve the viability of the neuronal cell culture. in relation to the culture prior to providing the hematopoietic factor. In said method, the polypeptide of the present invention can be used to contact the cells of the culture or can be provided by the use of polynucleotides that encode and express the polypeptide of the present invention.

The present application further describes a method for treating a neurological condition such as trinucleotide recurrence disorder, peripheral neuropathy, lysosomal storage disease, parkinsonism or glaucoma with a polynucleotide of the present invention or its fusion protein.

A method for improving the interaction between the CSF3R and its ligand in a subject is disclosed herein, the method comprises administering to the subject an effective amount of a product as defined above.

A method for reducing the interaction between CSF3R and its ligand in a subject is further disclosed, the method comprises administering to the subject an effective amount of a product as defined above.

The term treatment includes preventive or curative treatments in a subject, particularly a human subject. Treatment includes any improvement of a clinical manifestation of a disease, which delays the onset of the disease, particularly the onset of an acute disease, reduces its severity and reduces the evolution of the disease or suppresses its causes.

Effective doses can be adjusted by the person skilled in the art, depending on the patient, disease and product. Generally, effective doses comprise between about 5 μg / kg and 50 mg / kg, particularly between 100 μg / kg and 10 mg / kg.

The pharmaceutical compositions may contain one or more products of the present invention, either only as active ingredients or for use in combination with other active ingredients and any biologically compatible diluent, vehicle, vehicle and suitable pharmaceutically acceptable additive, which are suitable for administration to an animal (for example, physiological saline) and optionally comprise auxiliaries (such as excipients, stabilizers or adjuvants) that facilitate the processing of active compounds in preparations that can be used for pharmaceutical use. The pharmaceutical compositions can be formulated in any acceptable way to meet the needs of the mode of administration. For example, the use of biomaterials and other polymers for drug administration is disclosed in the literature, as well as the different techniques and models to validate a specific mode of administration (Luo B and Prestwich GD, 2001; Cleland JL et al., 2001).

The term "pharmaceutically acceptable" is intended to encompass any vehicle, which does not interfere with the efficacy of the biological activity of the active ingredient and which is not toxic to the host to which it is administered. For example, for parenteral administration, the above active ingredients can be formulated in unit dosage form for injection into vehicles such as saline, dextrose solution, serum albumin and Ringer's solution. Vegetables can be selected from starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, crete, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dehydrated skim milk , glycerol, propylene glycol, water, ethanol and the different oils, which include those of petroleum or animal, vegetable or synthetic origin (peanut oil, soybean oil, mineral oil, sesame oil).

Any accepted mode of administration can be used and determined by those skilled in the art to establish the desired blood levels of the active ingredients. For example, administration can be by parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, rectal, oral or oral routes. The pharmaceutical compositions of the present invention may also be administered in controlled or sustained release dosage forms, including injection with gradual release of the medication, osmotic pumps and the like, for prolonged administration of the polypeptide at a predetermined rate, preferably in forms. of unit dosage suitable for unit administration of precise doses.

Parenteral administration can be by bolus injection or by gradual infusion over time. Preparations for parenteral administration include aqueous or non-aqueous sterile suspensions and emulsions solutions, which may contain auxiliary agents or excipients known in the art and may be prepared in accordance with routine methods. Likewise, suspensions of active compounds can be administered as suitable oily suspensions. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil or esters of synthetic fatty acids, for example, ethyl oleate or triglycerides. Aqueous injection suspensions that may contain substances that increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol and / or dextran. Optionally, the suspension may also contain stabilizers. The pharmaceutical compositions include solutions suitable for administration by injection and contain from about 0.01 to 99.99 percent, preferably from about 20 to 75 percent of the active compound together with the excipient.

It is understood that the dose administered will depend on the age, sex, health status and weight of the recipient, type of concurrent treatment, if any, frequency of treatment and the nature of the desired effect. The dose will be adapted to the particular subject, as understood and determined by the person skilled in the art. The total dose required for each treatment can be administered by multiple doses or in a unit dose. The pharmaceutical composition of the present invention can be administered alone or together with other therapeutic products directed to the condition or directed to other symptoms of the condition. Usually a daily dose of the active ingredient comprises between 0.01 to 100 milligrams per kilogram of body weight per day. Generally, administration of 1 to 40 milligrams per kilogram per day in divided doses or in the form of sustained release is effective in obtaining the desired results. The second or subsequent administrations may be performed at a dose that is equal to, less than or greater than the initial or previous dose administered to the individual.

The present application further describes compositions and methods for detecting or dosing a polypeptide or nucleic acid of the present invention in a sample. Such compositions include, for example, any specific ligand of a polypeptide of the present invention, such as an antibody or its fragment or derivative or any specific nucleic acid probe or primer.

An antibody or its fragment or derivative that selectively binds to a variant of CSF3R polypeptide as disclosed above is also disclosed. The antibody, its fragment or derivative, binds selectively to an epitope of the polypeptides of the present invention. It was found that residues His 4, Fe 74, Gln 86, Leu 88 and Gln 90 of SEQ ID NO: 4 are important for the receptor function, with Gln 87 being the most important (Layton et al.). Therefore, preferred epitopes consist of epitopes comprising these residues, in particular Gln 87, Fe 74, Gln 86 and Gln 90 of SEQ ID NO: 4. These residues are found in the β strands of F and Gdel CSF3R fixed . In Layton et al. An LMM711 mAb is also disclosed, which was also found in the F and G strands of the Ig type domain, superimposed on the CSF3 binding site, in accordance with the strong neutralizing effect of this mAb. Therefore, a preferred epitope is one comprising Fe 74, Gln 86 and Gln 90 of SEQ ID NO: 4. A pharmaceutical composition comprising an antibody, its fragment or derivative as defined above is also disclosed.

Preferred fragments of the Ig type domain comprise or consist of His 4, Fe 74, Gln 86, Leu 88 and Gln 90 residues of SEQ ID NO: 4. The most preferred fragments of the Ig type domain comprise or consist of Fe residues 74, Gln 86 and Gln 90 of SEQ ID NO: 4. Alternatively, preferred fragments of the Ig type domain comprise or consist of the F and G strands of the Ig type domain.

Within the context of the present invention, the term "selective" binding indicates that antibodies preferentially bind to the white polypeptide or epitope, that is, with an affinity greater than any link to any other antigen or epitope. In other words, binding to the white polypeptide can be discriminated from non-specific binding to other antigens.

The antibodies can be coupled to heterologous portions, such as toxins, labels, drugs or other therapeutic agents, covalently or not, either directly or through the use of coupling agents or linkers.

The antibodies of the present invention can be used to detect, dose, purify or neutralize the CSF3R polypeptide variant of the present invention. A method for detecting or dosing a variant of CSF3R polypeptide as defined above in a sample, comprising contacting said sample in contact with an antibody, its fragment or derivative as disclosed above, and determining formation or dosage is disclosed herein. the (relative) amount of an immune complex. The sample can be for example any biological fluid such as blood, plasma, serum, etc., optionally diluted and / or treated. The antibody, its fragment or derivative can be in suspension or immobilized on a support. The presence or amount of immune complexes can be determined by any technique known per se in the art, for example, by ELISA, RIA, etc., for example, by the use of reporter antibodies, labeled antibodies, etc.

Methods for producing antibodies, their fragments or derivatives are well known in the art and include immunization of an animal and the collection of serum (polyclonal) or spleen cells (to produce hybridomas by fusion with suitable cell lines). Methods for producing polyclonal antibodies of various species, including rodents, primates and horses, were described, for example, in Vaitukaitis et al. (J Clin Endocrinol Metab. 33 (1971) p. 988). In summary, the antigen is combined with an adjuvant (for example, Freund's adjuvant) and is administered to an animal, generally by subcutaneous injection. Repeated injections can be applied. Blood samples are collected and immunoglobulins or serum are separated. The monoclonal antibodies of the CSF3R polypeptides of the invention can also be easily produced by one skilled in the art. The general methodology for preparing monoclonal antibodies by using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Antibodies: A laboratory Manual, CSH Press, 1988; Kohler et al Nature 256 ( 1975) 495). In summary, these methods comprise the immunization of an animal with the antigen and then the recovery of the spleen cells and the fusion of these cells with immortalized cells, such as myeloma cells to produce hybridomas. The hybridomas that produce the desired monoclonal antibodies can be selected by limit dilutions. Antibodies can also be produced by the selection of combinatorial immunoglobulin libraries, as disclosed for example in Ward et al (Nature 341 (1989) 544).

Monoclonal antibody panels produced against the polypeptides of the first aspect of the invention can be analyzed to identify various properties, that is, identify isotypes, epitopes, affinity, etc. Monoclonal antibodies are particularly useful for the purification of the individual polypeptides against which they are directed. Alternatively, the genes encoding the monoclonal antibodies of interest can be isolated from hybridomas, for example by PCR techniques known in the art and can be cloned and expressed in suitable vectors.

The chimeric antibodies in which non-human variable regions are bound or fused to human constant regions can also be used (see, for example, Liu et al., Proc. Natl. Acad. Sci. USA, 84, 3439 (1987) ).

The antibody can be modified to become less immunogenic in an individual, for example by humanization (see Jones et al., Nature, 321, 522 (1986); Verhoeyen et al., Science, 239, 1534 (1988); Kabat et al. , J. Immunol., 147, 1709 (1991); Queen et al., Proc. Natl. Acad. Sci. USA, 86, 10029 (1989); Gorman et al., Proc. Natl. Acad. Sci. USA, 88, 34181 (1991); and Hodgson et al., Bio / Technology, 9, 421 (1991)). The term "humanized antibody", as used herein, refers to antibody molecules in which the CDR amino acids and other amino acids selected in the variable domains of the heavy and / or light chains of an antibody from a non-donor donor human were substituted instead of the equivalent amino acids in a human antibody. The humanized antibody therefore closely resembles a human antibody but has the ability of donor antibody binding.

The antibody can be a "bispecific" antibody, that is, an antibody that has two different antigen-binding domains, each domain directed to a different epitope.

Phage expression technology can be used to select genes encoding antibodies with binding activities to the polypeptides of the invention either from repertoires of V genes amplified by PCR from human lymphocytes selected for possessing the relevant antibodies, or from simple libraries ( McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain transpositioning (Clackson, T. et al., (1991) Nature 352, 624628).

Antibodies generated by the foregoing techniques, either polyclonal or monoclonal, have an additional utility since they can be used as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunoabsorbent assays (ELISA). In these applications, the antibodies can be labeled with an antianalytically detectable reagent as a radioisotype, a fluorescent molecule or an enzyme.

Generally, polyclonal antibodies directed towards a polypeptide of the present invention are produced in animals (eg, rabbits or mice) by subcutaneous or intraperitoneal injections of the sCSF3R polypeptide and an adjuvant. It may be useful to conjugate a polypeptide of the present invention for a transporter protein that is immunogenic in the species to be immunized, such as hemocyanin from Californian lapa, serum, albumin, bovine thyroglobulin or soy trypsin inhibitor. Also, aggregating agents such as alum are used to improve the immune response. After immunization, blood is drawn from the animals and the serum is analyzed for titration of the anti-sCSF3R antibody.

Monoclonal antibodies directed to a polypeptide of the present invention are produced by the use of any method intended for the production of antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies include the hybridoma methods and the human B cell hybridoma method. Hybridoma cell lines producing monoclonal antibodies with sCSF3R polypeptides are also disclosed herein.

The monoclonal antibodies of the invention can be modified for use as therapeutic products. One embodiment is a "chimeric" antibody in which a portion of the heavy (H) and / or light (L) chain is identical or homologous to a corresponding sequence of antibodies derived from a particular species or belonging to a class or subclass particular antibody, while the rest of the chain or chains is identical or homologous to a corresponding sequence of antibodies of another species or belonging to another class or subclass of antibodies. Fragments of said antibodies are also included provided they demonstrate the desired biological activity.

A monoclonal antibody that is a "humanized" antibody is also disclosed. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. Humanization can be carried out, for example, by using methods described in the art, by replacing at least a portion of a rodent complementarity determining region for the corresponding regions of a human antibody.

The term "antibody" or "immunoglobulin" is intended to encompass polyclonal and monoclonal antibodies. The preferred antibody is a monoclonal antibody reactive with the antigen. Likewise, the term "antibody" is intended to encompass mixtures of more than one antibody reactive with an antigen (for example, a cocktail of different types of monoclonal antibodies reactive with an antigen). The term "antibody" is also intended to encompass complete antibodies, their biologically functional fragments, single chain antibodies and genetically altered antibodies such as chimeric antibodies comprising portions of more than one species, bifunctional antibodies, antibody conjugates, humanized and non-humanized antibodies. Biologically functional antibody fragments that can also be used are those fragments of peptides derived from an antibody that are sufficient to bind to the antigen. The antibody as used herein is intended to include the complete antibody as well as any antibody fragment (eg, F (ab ′) 2, Fab ′, Fab, Fv) capable of binding to the epitope, antigen or antigenic fragment of interest.

The term "purified antibody" refers to one that is sufficiently free of other proteins, carbohydrates and lipids with which they are naturally associated. Said antibody "preferentially binds" to the sCSF3R polypeptides of the present invention (or its antigenic fragment), that is, does not recognize and does not bind to other unrelated antigenic molecules. A purified antibody that is disclosed herein preferably immunoreactive and immunospecific of sCSF3R of specific species and more preferably immunospecific for native human sCSF3R.

The term "specifically binds" refers to a high avidity and / or high affinity linkage of an antibody to a specific polypeptide, ie the sCSF3R. The binding of the antibody to its epitope on this specific polypeptide is preferably stronger than the binding of the same antibody to any other epitope. Antibodies that specifically bind to sCSF3R may be able to bind to other polypeptides at a weak yet detectable level (e.g., 10% or less of the link shown to the polypeptide of interest). Such weak bond or background bond can be readily discerned from the specific antibody that binds to the compound or polypeptide of interest, for example, by the use of suitable controls.

Preferably, the affinity is at least 1.5 times, 2 times, 5 times, 10 times, 100 times, 10 3 times, 10 4 times, 10 5 times or 10 6 times greater for a polypeptide of the invention than for others known members of the CSF3R family.

The term "genetically altered antibodies" means antibodies where the amino acid sequence was changed with respect to that of the native antibody. Due to the relevance of recombinant DNA techniques for the present invention, one should not be limited to the amino acid sequences found in natural antibodies; The antibodies can be redesigned to obtain the desired characteristics. The possible variations are many and vary from the change of only one or several amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region will be made, in general, to improve or alter its characteristics, such as complementary fixation, interaction with membranes and other effector functions. Changes in the variable region will be made to improve the binding characteristics of the antigen.

The term "humanized antibody" or "humanized immunoglobulin" refers to an immunoglobulin comprising a human framework, at least one and preferably all complementarity determining regions (CDR) of a non-human antibody and in which any constant region present is substantially identical to a constant region of human immunoglobulin, that is, at least about 85-90%, preferably at least 95% identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of one or more native human immunoglobulin sequences. See, for example, Queen et al., United States patents Nos. 5,5301,101; 5,585,089; 5,693,762 and 6,180,370.

"Fully humanized antibodies" are molecules that contain both the constant region and the variable region of human immunoglobulin. Fully humanized antibodies can potentially be used for therapeutic use, where repeated treatments are required for chronic and recurrent diseases such as autoimmune diseases. A method for the preparation of fully human antibodies consists in the "humanization" of the mouse humoral immune system, that is, the production of mouse strains capable of producing human Ig (Xenomice), by the introduction of human immunoglobulin (Ig) in the mouse where the endogenous Ig genes were activated. The regions of Ig are excessively complex in terms of both their physical structure and gene reorganization and the expression processes required to ultimately produce a broad immune response. Antibody diversity is mainly generated by combinatorial reorganization between the different V, D and J genes present in the Ig regions. These regions also contain intercalated regulatory elements that control antibody expression, allelic exclusion, class change and affinity maturation. The introduction of unordered human Ig transgenes in mice has shown that the mechanism of mouse recombination is compatible with human genes. Likewise, hybridomas that secrete specific hu-mAbs of antigens of various isotypes can be obtained by Xenomice immunization with antigens.

Fully humanized antibodies and methods for their production are well known in the art (Mendez et al., Nature Genetics 15: 146-156 (1997); Buggemann et al., Eur. J. Immunol. 21: 1323-1326 ( 1991); Tomizuka et al., Proc. Natl. Acad. Sci. United States 97: 722-727 (2000) Patent WO 98/24893.

The term "chimeric antibody" refers to an antibody in which the constant region comes from an antibody of one species (usually a human) and the variable region comes from an antibody of another species (usually a rodent). Thus, chimeric antibodies are molecules from which different portions are derived from different animal species, such as those in which their variable region is derived from a murine Mab and a constant region of human immunoglobulin. Chimeric antibodies are mainly used to reduce immunogenicity in the application and to increase production yields, for example, where murine Mabs have a higher yield in hybridomas but a greater immunogenicity in humans, such that Mabs are used human / murine chimerics. Chimeric antibodies and methods for their production are well known in the art (Cabilly et al., Proc. Natl. Acad. Sci. USA 81: 3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984); Boulianne et al., Nature 312: 643-646 (1984); Cabilly et al., European Patent Application 125023 (published November 14, 1984); Neuberger et al. ., Nature 314: 268-270 (1985); Taniguchi et al., European Patent Application 171496 (published February 9, 1985); Morrison et al., European Patent Application 173494 (published March 5, 1986 ); Neuberger et al., PCT application WO 8601533, (published March 13, 1986); Kudo et al., European patent application 184187 (published June 11, 1986); Sahagan et al., J. Immunol 137: 1066-1074 (1986); Robinson et al., International Patent Application No. WO8702671 (published May 7, 1987); Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439- 3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218 (1987); Better et al., Science 240: 1041-1043 (1988); Riechmann et al., Nature 332: 323-327. and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra.

As used herein, the phrase "antibody fragment" refers to a molecule comprising a portion of an antibody capable of specifically binding to an antigen, an antigenic determinant or an epitope. It will be noted that Fab and F (ab ′) 2 fragments and other antibody fragments useful in the present invention can be used for the detection and quantification of their antigens in accordance with the methods disclosed herein for intact antibody molecules. Such fragments are generally produced by proteolytic cleavage, by the use of enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ′) 2 fragments).

With regard to the antibodies mentioned herein, the term " monoclonal antibody " It is intended to include monoclonal antibodies, chimeric antibodies, fully humanized antibodies, antibodies to anti-idiotypic antibodies (anti-anti-Id antibody) that can be labeled in soluble or bound form, as well as their fragments provided by any known technique, such as not Taxative, enzymatic cleavage, peptide synthesis or recombinant techniques. A monoclonal antibody contains a substantially homogeneous population of antigen-specific antibodies, with populations containing substantially similar epitope binding sites. Mabs can be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature, 256: 495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al., Eds., Harlow and Lane ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience N.Y., (1992-1996). Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, GILD and any of its subclasses. The hybridoma that produces a Mab of the present invention can be cultured in vitro, in situ or in vivo. The production of high Mabs values in vivo or in situ makes this the preferred production method. Likewise, the term "monoclonal antibody" is intended to include both intact molecules and their fragments, such as, for example, Fab and F (ab ′) 2 that are capable of binding to the antigen. Fab and F (ab ′) 2 fragments lack the intact antibody Fc fragment, leave faster circulation and may have less specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24: 316 -325 (1983)).

A monoclonal antibody is said to be "capable of binding" to a molecule if it is able to react specifically with the molecule to thereby bind the molecule to the antibody.

An "antigen" is a molecule or a portion of a molecule capable of binding by an antigen which is also capable of causing an animal to produce antibodies capable of binding to an epitope of said antigen. An antigen can have one or more of an epitope. The specific reaction referred to above is intended to indicate that the antigen will react, highly selectively, with an epitope on its corresponding antibody and not with a multitude of other antibodies that may be raised by other antigens.

The antibodies, including the antibody fragments disclosed herein can be used to quantitatively or qualitatively detect their antigens in a sample or to detect the presence of cells expressing their antigens. This can be achieved by immunofluorescence techniques by using fluorescently labeled antibodies (see below) coupled with fluorescence microscopy, flow cytometry.

or fluorometric detection.

The antibodies (or their fragments) disclosed herein can be used histologically, as in immunofluorescence or immunoelectronic microscopy for in situ detection of their antigens. In situ detection can be achieved by extracting the histological samples from a patient and supplying the labeled antibody of the present invention to said sample. The antibody (or its fragment) is preferably provided by the application or coating of the labeled antibody (or its fragment) on a biological sample. By using such a procedure, it is possible to determine not only the presence of the antigens but also their distribution in the examined tissue. Those skilled in the art will readily perceive that any of the wide variety of histological methods (such as staining procedures) can be modified to achieve such detection in situ.

Such assays for the antigens generally comprise incubating a biological sample, such as a biological fluid, a tissue extract, newly collected cells such as lymphocytes or leukocytes or cells that were incubated in tissue culture, in the presence of a labeled antibody capable of identifying the antigens and to detect the antibody by any of a variety of techniques known in the art.

The biological sample can be coupled to a solid phase carrier or vehicle such as nitrocellulose or other solid carrier or vehicle that is capable of immobilizing cells, cell particles or soluble proteins. The carrier or vehicle can then be washed with suitable buffer solutions followed by treatment with an antibody labeled in accordance with the present invention as noted above. The solid phase carrier or vehicle can then be washed with the buffer solution a second time to extract unbound antibody. The amount of tag bound on said support or solid vehicle can then be detected by conventional means.

The antibody molecule can be adapted for use in an immunometric assay, also known as a "double phase" assay. In a typical immunometric assay, an amount of unlabeled antibody (or antibody fragment) binds to a solid carrier or carrier and an amount of tagged soluble antibody is added to allow detection and / or quantification of the ternary complex formed between the solid phase antibody, the antigen and the labeled antibody.

Antibodies can be used in relation to immunoaffinity chromatography technology. More specifically, antibodies can be placed on the surface of a material within column chromatography. Then, the composition to be purified can be passed through the column. If the sample to be purified includes any sCSF3R polypeptide that binds to the antibodies, the sCSF3R polypeptides will be extracted from the sample and thereby purified.

Thus, in summary, diagnostic methods can be performed in vitro by using a cell sample (eg, blood sample, biopsy or lymph node tissue) of a mammal or can be performed by in vivo imaging.

Compositions comprising antibodies disclosed herein can be used to detect the presence of sCSF3R, for example, by radioimmunoassay, ELISA, FACS, etc. One or more marker portions may bind to humanized immunoglobulin. Exemplary labeled portions include radiopaque dyes, radiocontrast agents, fluorescent molecules, rotationally labeled molecules, enzymes or other labeled portions of diagnostic value, particularly in radiological or magnetic resonance imaging techniques.

An IgG antibody preparation can be advantageously purified from an anti-serum disclosed herein by using affinity purification with G protein, preferably by immunoprecipitation with G protein. An anti-serum derived from an animal can be used. immunized to detect with optimal sensitivity, by immunoblot analysis (Western blot), immunoprecipitation and ELISA, the sCSF3R polypeptides.

In general, for applications that benefit from optimal reproducibility, standardization or precision, a purified antibody or antibody fragment of the present invention capable of specifically binding to the white antigen will generally be optimal in relation to an unpurified preparation.

Those skilled in the art will appreciate that an antibody or antibody fragment having an affinity characterized by a dissociation constant of up to 10-12 for a cognate antigen can be obtained through the use of methods common in the art.

The preparation may advantageously comprise an antibody or an antibody fragment bound to any of several types of detectable molecules.

An antibody fragment has the advantage of being smaller than the parent antibody from which it is derived while retaining a binding specificity to the white antigen or both a binding specificity and binding affinity substantially identical to that of the parent antibody. Therefore, since the antibody fragment is smaller than the parent antibody, it will generally have a biodistribution and diffusion properties (for example, systemically in vivo or in isolated tissues) superior to those of the latter. An antibody fragment that substantially lacks an Fc region, such as a single chain Fv, a Fab ′, a Fab, an F (ab ′) 2 and a CDR, is advantageous for applications that involve exposure of the preparation of a molecule capable of specifically binding to said Fc region and whose binding is undesirable. Generally this may involve an unwanted binding of an Fc region exposed to a cognate Fc receptor or a complementary Fc binding component (for example, a complementary Clq component present in the serum). Fc receptors occur on the surface of numerous types of immune cells that include: professional APCs, such as dendritic cells, B lymphocytes and granulocytes such as neutrophils, basophils, eosinophils, monocytes, macrophages and mast cells. Therefore, the absence of an Fc region of the antibody fragment can be particularly advantageous in preventing the activation of immune cells mediated by the Fc receptor or the complementary cascade mediated by the complementary component, particularly when administering the preparation in vivo to an individual.

F (ab ′) 2 is a fragment of an antibody molecule that contains a divalent antigen binding portion of an antibody molecule.

The preparation of F (ab ′) 2 can be conveniently obtained by the use of standard methods in the art by treating an antibody preparation, such as an anti-serum of the present invention with the enzyme pepsin. The resulting F (ab ′) 2 product is a 5S particle.

F (ab ′) 2 or Fab ′ is a fragment of an antibody molecule that contains a monovalent antigen binding portion of an antibody.

The CDR can be generated, for example, as described in EP0585939 or as described by Strandberg et al. (Protein Eng. January 2001; 14 (1): 67-74). The CDR according to the invention can be a modified CDR that has an improved effect on the modulation of the sCSF3R polypeptide. An example of methods of modifying active peptides is described in Sawa et al. 1999 (J. Med. Chem. 42, 3289-3299).

The preparation of Fab ′ can be conveniently obtained by the use of standard methods in the art through the treatment of an antibody preparation of the present invention, as an anti-serum of the present invention, with the enzyme pepsin followed by the reduction of the resulting F (ab ′) 2. Such reduction can be accomplished by the use of a thiol reducing agent and optionally by the use of a blocking group for sulfhydryl groups resulting from the cleavage of the disulfide bonds. This treatment generates two monovalent 3,5S Fab's and an Fc fragment.

Fab preparation can be conveniently obtained by using standard methods in the art through the treatment of an antibody preparation, such as an anti-serum, with the papain enzyme to produce the intact light chain and a heavy chain portion. composed of the variable domains and CH 1.

A wide variety of instructions for generating an antibody fragment by enzymatic treatment of an antibody are provided in the literature of the art (eg, refer to: Goldenberg, US Pat. Nos. 4,036,945 and 4,331,647; Porter R. R. , 1959. Biochem J. 73: 119-126).

A single chain Fv (also referred to in the art as "scFv") is a single chain molecule that includes the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker.

The preparation of the single chain F (ab ′) 2, Fab ′, Fab or Fv or of the CDR can be obtained through the use of recombinant techniques.

Obtaining a recombinant antibody fragment is carried out by isolating the mRNA from B lymphocytes from animals immunized with the white antigen, which generates cDNA from the mRNA by RT-PCR and by using cDNA to construct a phage expression library of antibody fragments. B lymphocytes can be conveniently isolated from the spleen or alternatively, from the blood, bone marrow or lymph nodes of the immunized animal.

It will be appreciated that the methodology described above can be used to obtain a preparation of a monoclonal antibody fragment of the present invention having essentially any affinity and / or specificity of binding to the desired white antigen. Such a preparation can be used in several applications that benefit from a reagent capable of binding to the white antigen with said defined characteristics of binding to the white antigen.

Because the Fab ′ is essentially similar to the Fab in structure, a preparation comprising a Fab ′ can be used basically interchangeably with one comprising a Fab, where said Fab ′ and Fab essentially comprise the same light chain variable regions and heavy For applications, as will generally be the case, which benefit from a preparation comprising an antibody fragment capable of binding to the white antigen with a maximum affinity, a preparation of F (ab2 may be greater than a preparation

′) Tion of Fab, Fab ′ or scFv due to the divalent bond of F (ab ′) 2 to the white antigen in relation to the monovalent bond of said monovalent antibody fragment.

As mentioned above, depending on the application and purpose, the preparation of antibody or antibody fragment can originate from several mammalian species.

An antibody or antibody fragment preparation that originates from a desired species can be derived from the serum of the animal of said species immunized with the white antigen.

A preparation of a human or humanized antibody or antibody fragment may be preferred for applications that involve administration of the preparation to an individual. For example, a human or humanized antibody or an antibody fragment will generally tend to be optimally immunologically tolerated and therefore will show an excellent half-life in vivo in a human and thereby show optimum efficacy. Additional instructions regarding the production and exploitation of human or humanized antibodies are provided below.

The preparation can be used by itself or can be formulated as an active ingredient in a pharmaceutical composition.

Therefore, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient an antibody or an antibody fragment is disclosed.

The methods for formulating the antibody or antibody fragment as an active ingredient in a pharmaceutical composition and the methods for exploiting said pharmaceutical composition are described below.

Preferably, the administration of the antibody or antibody fragment is accomplished by the administration of the pharmaceutical composition disclosed herein comprising the antibody or antibody fragment as active ingredient.

The antibody or antibody fragment is preferably administered in such a way as to achieve a sufficient level of antibody fragment bound to the white antigen to achieve a desired regulation of biochemical activity.

A person skilled in the art, such as a doctor, more preferably a doctor specialized in the disease, will have the experience required to determine a suitable therapeutic protocol, which includes a suitable route of administration and an adequate dose of antibody or antibody fragment to treat the disease. disease effectively.

The present invention further describes a nucleic acid probe, wherein said probe selectively hybridizes to a nucleic acid as defined above or its complementary strand. The probes denote a defined segment of nucleic acid (or nucleotide analog segment, for example, a polynucleotic as defined herein) that can be used to identify a specific sequence of polynucleotides present in the samples, said nucleic acid segment comprises a nucleotide sequence that complements the specific polynucleotide sequence to be identified. The probes of the present invention generally comprise single chain nucleic acids between 12 to 600 nucleotides in length, for example between 12 and 500, more preferably between 15 and 400, generally between 20 and 300. The sequence of the probes can be derived from the sequences of the gene sequence of the polypeptide variant of the CSF3R. The probe may contain nucleotide substitutions and / or chemical modifications, for example, to increase the stability of the hybrids or to label the probe. Typical examples of labels include, by way of non-taxation, radioactivity, fluorescence, luminescence, etc.

A nucleic acid primer that can be used to amplify at least one characteristic fragment of a nucleic acid molecule encoding a variant of CSF3R polypeptide as defined above is also described. A "primer" means a specific sequence of oligonucleotides that complements a white nucleotide sequence and is used to hybridize the white nucleotide sequence. The primer serves as a starting point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase or reverse transcriptase. Typical primers of the present invention are single chain nucleic acid molecules of about 6 to 50 nucleotides in length, more preferably about 8 to about 40 nucleotides in length. The primer sequence can be derived directly from the sequence of the white nucleic acid molecule. Perfect complementarity between the primer sequence and the white gene is preferred to ensure high specificity. However, certain mismatches can be tolerated.

Particular nucleic acid primers are capable of hybridizing specifically with a nucleic acid portion of the CSF3R variant that flanks or encodes a characteristic fragment of said polypeptides. Specific examples of primers of the present invention are disclosed below:

sCSF3R-F1: ATGGCAAGGCTGGGAAACTG (SEQ ID NO: 13) sCSF3R-R1: TTTAGTAGAGGCGGGGTTTCG (SEQ ID NO: 14)

nested sCSF3R-F1: ACTTGGGCTGCCCTGATCATCC (SEQ ID NO: 15) nested sCSF3R-R1: TGGCCAGGCTGATCATGAACTC (SEQ ID NO: 16)

sCSF3R-EX1: GAAACTGCAGCCTGACTTGGGCTGCCCTGATCA (SEQ ID NO: 17) sCSF3R-EX2: GAGGCGGGGTTTCGCCTTGCTGGCCAGGCTGATCATGAAC (SEQ ID NO: 18) sCSF3R-EX3: ATGGCAAGGCTGGGAAACTGCAGCCTGACTT (SEQ ID NO: 19) sCSF3R-EX4: ATTTTTTTGTATTTTTAGTAGAGGCGGGGTTTCGCCTT (SEQ ID NO: 20) sCSF3R- EX5: GCAGGCTTCGCCACCATGGCAAGGCTGGGAAACTG (SEQ ID NO: 21) sCSF3R-EX6: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCACC (SEQ ID NO: 22)

The use of a primer or probe as disclosed above to detect or diagnose the presence of a nucleic acid encoding a polypeptide variant of the CSF3R of the present invention in a sample is also described. The method can be carried out in accordance with techniques well known in the art, such as contacting a sample with a probe as defined above under conditions that allow hybridization to occur and determining the presence of a hybrid, or contacting a Sample with a primer as defined above under conditions that allow nucleic acid amplification and determine the presence of an amplification product.

The use of nucleic acid molecules in accordance with the present invention as diagnostic reagents is also disclosed. The detection of a mutant form of the gene characterized by the nucleic acid molecules of the invention that is associated with a dysfunction will provide a diagnostic tool that can aggregate or define the diagnosis of a disease or susceptibility to a disease, resulting from the under-expression, overexpression or altered spatial or temporal expression of the gene. Individuals with mutations in the gene can be detected at the DNA level by a variety of techniques.

Nucleic acid molecules for diagnosis can be obtained from the cells of a subject, such as blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA can be used directly for detection or can be amplified enzymatically by the use of PCR, ligase chain reaction (CSF), strand displacement amplification (SDA) or other amplification techniques (see Saiki et al., Nature, 324, 163166 (1986); Bej, et al., Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al., J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio / Technology, 8, 291-294 (1990)) before analysis.

A method for diagnosing a disease in a patient comprising assessing the level of expression of a natural gene encoding a polypeptide in accordance with the invention and comparing said level of expression with a control level is disclosed herein, where the level that different from that level of control represents an indication of the disease. This method can comprise the following stages:

a) contacting a tissue sample of the patient presenting a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the present invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); c) and detect the presence of hybrid complexes in said samples; where the detection of levels of the hybrid complex in the patient sample that differ from the levels of the hybrid complex in the control sample represents an indication of the disease.

A diagnostic method is also described which comprises the following stages:

a) obtain a tissue sample from a patient who was examined for the disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and c) diagnose the disease in a patient by detecting the presence of mutation in the nucleic acid molecule associated with the disease.

To aid in the detection of nucleic acid molecules in the methods described above, an amplification step may be included, for example by the use of PCR.

Eliminations and insertions can be detected by changing the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to the labeled RNA of the invention or alternatively, to labeled antisense DNA sequences of the present invention. Perfectly matched sequences can be distinguished from unpaired duplexes by digesting RNase or by evaluating differences in melting temperatures. The presence or absence of mutation in a patient can be detected by contacting the DNA with a nucleic acid probe that hybridizes to the DNA under stringent conditions to form a hybrid double chain molecule, the hybrid double chain molecule has a portion without hybridize to the nucleic acid probe chain in any portion that corresponds to a mutation associated with the disease; and presence detection

or absence of an unhybridized portion of the probe chain indicates the presence or absence of a mutation associated with the disease in the corresponding portion of the DNA chain.

Such diagnoses are particularly useful for prenatal and even neonatal tests.

Point mutations and other sequence differences between the reference gene and the "mutant" genes can be identified by other methods well known in the art, such as direct DNA sequencing or single-chain conformational polymorphism (see Orita et al., Genomics, 5, 874-879 (1989)). For example, a sequencer primer can be used with the product of the double chain PCR or with a single chain matrix molecule generated by a modified PCR. Sequence determination is done by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent labels. Cloned segments of DNA can also be used as probes to detect specific DNA segments. The sensitivity of this method greatly improves when combined with PCR. Likewise, point mutations and other sequence variations such as polymorphisms can be detected as described above, for example, by the use of allele-specific oligonucleotides for PCR amplification of sequences that differ in single nucleotides.

Differences in DNA sequences can also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al., Science (1985) 230: 1242). Changes in sequences in specific locations can also be revealed by nuclease protection assays, such as RNase and SI protection or chemical cleavage method (see Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).

In addition to conventional gel electrophoresis and DNA sequencing, mutations such as microeliminations, aneuploidies, displacements, inversions, can also be detected by in situ analysis (see, for example, Keller et al., DNA Probes, 2nd Ed., Stockton Press, New York, NY, USA (1993)), that is, DNA or RNA sequences in cells can be analyzed for mutations without the need for their isolation and / or immobilization in a membrane. Fluorescent in situ hybridization (FISH) is currently the most commonly used method and numerous reviews of FISH have been published (see, for example, Trachuck et al., Science, 250, 559-562 (1990), and Trask et al ., Trends, Genet., 7, 149-154 (1991)).

A network of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to carry out an efficient analysis of genetic variants, mutations and polymorphisms. Network technology methods are well known in the art and have a general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage and genetic variability (see for example: M. Chee et al., Science (1996), Vol 274, pp 610-613).

A network prepared and used in accordance with the methods described in PCT application WO95 / 11995 (Chee et al) is also disclosed; Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al. (1996) Proc. Natl Acad. Sci. 93: 10614-10619). The oligonucleotide pairs can vary from two to more than one million. The oligomers are synthesized in designated areas on a substrate that employs a light-directed chemical process. The substrate can be paper, nylon or other type of membrane, filter, chip, glass or any other suitable support slide. The oligonucleotide can be synthesized on the surface of the substrate by the use of a chemical coupling method and an ink jet application apparatus as described in PCT application WO95 / 25116 (Baldeschweiler et al). A "crosslinked" network analogous to a point transfer can be used to organize and bond the cDNA fragments or oligonucleotides to the surface of a substrate using vacuum, thermal, UV, mechanical or chemical system bonding procedures. A network may be manufactured, such as those described above, by hand or through the use of available devices (point transfer devices), materials (any suitable solid support) and machines (including robotics instruments) or may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides or any other number between two and more than one million which makes it suitable for the effective use of commercially available instruments.

In addition to the methods discussed above, diseases can be diagnosed by methods comprising determining, from a sample of a subject, an abnormal increase or decrease in the level of polypeptide or mRNA. The increase or decrease in expression can be measured at the level of RNA by the use of methods well known in the art for the quantification of polynucleotides, such as, for example, the amplification of nucleic acids, for example, PCR, RT-PCR, RNase protection, immunoblotting (Northern blotting) and other hybridization methods.

Assay techniques that can be used to determine the levels of a polypeptide of the present invention in a sample of a host are well known to those skilled in the art and are discussed in more detail above (including radioimmunoassays, competitive binding assays, Western Blot analysis and ELISA assays). A diagnostic method is further described comprising the following steps: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex and (b) detecting said complex.

Protocols such as ELISA, RIA and FACS for measuring polypeptide levels can also provide a basis for the diagnosis of abnormal or abnormal levels of polypeptide expression. Standard or normal values for polypeptide expression are established through the combination of body fluids or cell extracts taken from normal, preferably human, mammalian individuals with an antibody to the polypeptide under conditions suitable for complex formation. The amount of the standard complex formation can be quantified by various methods such as by photometric means.

Antibodies that specifically bind a polypeptide of the invention can be used for the diagnosis of conditions or diseases characterized by the expression of the polypeptide or in assays to control patients being treated with polypeptides, nucleic acid molecules, ligands. and other composites

cough of the invention. Antibodies useful for diagnostic purposes can be prepared in the same manner as those described above for therapeutic products. Diagnostic assays for the polypeptide include methods that use the antibody and a tag to detect the polypeptide in body fluids or human cell or tissue extracts. The antibodies can be used with or without modification and can be labeled by binding either by covalent or non-covalent bonding with a reporter molecule. A wide variety of reporter molecules known in the art can be used, many of which are described above.

The amounts of polypeptide expressed in subject, control and tissue disease samples to which a biopsy was performed are compared with standard values. The deviation between the standard values and those of the subject establishes the parameters to diagnose the disease. Diagnostic assays can be used to distinguish between the absence, presence and excess expression of the polypeptide and to control the regulation of polypeptide levels during therapeutic intervention. Such trials can also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in the control of the treatment of an individual patient.

A diagnostic kit is also disclosed that includes:

(to)

a nucleic acid molecule of the present invention;

(b)

a polypeptide of the present invention; or

(C)

a ligand of the present invention.

The diagnostic kit may comprise a first container containing a nucleic acid probe that hybridizes under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule and instructions for using the probe and primers to facilitate the diagnosis of the disease. The kit may further comprise a third container that has an agent to digest unhybridized RNA.

Alternatively, the diagnostic kit may comprise a network of nucleic acid molecules, at least one of which may be a nucleic acid molecule in accordance with the invention.

To detect the polypeptide according to the invention, the diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.

These kits will be used for the diagnosis of a disease or susceptibility to a disease selected from graft versus host disease, cancer, neutropenia, neurological disorders or conditions, pneumonia, autoimmune diseases, hematological diseases, hematopoietic disorders, infectious diseases, inflammatory bowel disease and diabetes

The expression of a polypeptide of the present invention is also an effective marker for the evaluation of the degree of myelopoiesis and activity or for the prognosis of malignant disorders of myeloid lineages (eg, myeloblastic leukemia) or hematopoietic disorders (Iwasaki et to the.).

Other aspects and advantages of the present invention will be disclosed in the following examples, these should be considered for illustrative purposes only and not limit the scope of the present application.

TABLE 1

Amino acid Synonyms groups

Most preferred synonyms groups

Be

Gly, Wing, Being, Thr, Pro Thr, Be

Arg

Asn, Lys, Gln, Arg, His Arg, Lys, His

Leu

Phe, Ile, Val, Leu, Met Ile, Val, Leu, Met

Pro

Gly, Wing, Being, Thr, Pro

Pro

Thr

Gly, Wing, Being, Thr, Pro Thr, Be

To

Gly, Thr, Pro, Wing, Being Gly Wing

Val

Met, Phe, Ile, Leu, Val Met, Ile, Val, Leu

Gly

Wing, Thr, Pro, Be, Gly Gly Wing

Ile

Phe, Ile, Val, Leu, Met Ile, Val, Leu, Met

Phe

Trp, Phe, Tyr Tyr, Phe

Tyr

Trp, Phe, Tyr Phe, Tyr

Cys

Be, Thr, Cys

Cys

31

His

Asn, Lys, Gln, Arg, His Arg, Lys, His

Gln

Glu, Asn, Asp, Gln Asn, Gln

Asn

Glu, Asn, Asp, Gln Asn, Gln

Lys

Asn, Lys, Gln, Arg, His Arg, Lys, His

Asp

Glu, Asn, Asp, Gln Asp, Glu

Glu

Glu, Asn, Asp, Gln Asp, Glu

Met

Phe, Ile, Val, Leu, Met Ile, Val, Leu, Met

Trp

Trp, Phe, Tyr

Trp

TABLE 2

Amino acid

Synonyms Groups

Ser D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met (O), D-Met (O), L-Cys, D-Cys Arg D-Arg, Lys, D-Lys , homo-Arg, D-homo-Arg, Met, Ile, D-.Met,

D-Ile, Orn, D-Orn Leu D-Leu, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Pro D-Pro, LI-thioazolidine-4-carboxylic acid, Do L-1

Oxazolidine-4-carboxylic acid Thr D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met (O), D-Met (O),

Val, D-Val Ala D-Ala, Gly, Aib, B-Ala, Acp, L-Cys, D-Cys Val D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met, AdaA, AdaG Gly Ala, D-Ala, Pro, D-Pro, Aib, β-Ala, Acp Ile D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Phe D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans

3,4, or 5-phenylproline, AdaA, AdaG, cis-3,4, or 5

Phenylproline, Bpa, D-Bpa Tyr D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Cys D-Cys, S - Me - Cys, Met, D-Met, Thr, D- Thr Gln D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Asn D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Lys D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile,

D-Ile, Orn, D-Orn Asp D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Glu D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Met D-Met, S - Me - Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val

EXAMPLES

Example 1 - Cloning of the domain of type Ig C2 of sCSF3R

Introduction

The sCSF3R is a variant of the CSF3R gene (colony stimulating factor 3 receptor). The CSF3R gene is composed of 15 coding exons. There are 2 non-coding exons in UTR 5 ’. The sCSF3R is a 189 amino acid protein that contains a signal peptide (amino acids 1-25) and an Ig C2 domain of the Lep receptor (amino acids 22-111). It is identical to the wild-type N-terminal CSF3R (Q99062) in the first 120 amino acids (which extend from exon 3 to the 5 ′ end of exon 4) but deviates in exon 4 so that an end C is thus introduced different and a premature termination codon that is predicted to produce a truncated protein.

Two pairs of PCR primers (sCSF3R-F1 / sCSF3R-R1 and nested sCSF3R-F1 / sCSF3R-R1) (FIG. 1) were designed to act as nested pairs, of which the first pair constitutes the external pair. The nested sCSF3R-F1 / sCSF3R-R1-nested was designed to amplify a 500 bp product between exons 3 and 4 of the sCSF3R sequence. A clone was identified, amplified from the PS3 group containing the cDNA derived from the mixed RA synovial membrane, mixed osteoarthritis and pulmonary fibroblasts containing the sCSF3R sequence except for the 27 bp missing at the 5 ′ end and 40 bp missing at the 3 ′ end due to the position of the nested primers used for the PCR. This clone is pCR4-TOPO-sCSF3RF1 / R1anided. Missing nucleotides were added during cDNA subcloning in the Gateway expression vectors.

SCSF3R cloning

a) Preparation of human cDNA models

The first strand cDNA was prepared from a variety of human tissue RNA samples (Clontech, Stratagene, Ambion, Biochain Institute and internal preparations) by using Superscript II or SuperScript III RNase H - Reverse Transcriptase (Invitrogen) in accordance with the manufacturer's protocol.

For SuperScript II: Oligo primer (dT) 15 (1 μl to 500 μg / ml) (Promega), 2 μg of total human RNA, 1 μl of the 10 mM dNTP mixture (10 mM each of the dATPs) was combined , dGTP, dCTP and dTTP at neutral pH) and sterile distilled water at a final volume of 12 μl, in an Eppendorf tube and heated at 65 ° C for 5 minutes and cooled with ice. The contents were collected by brief centrifugation and 4 μl of 5 × first strand buffer solution, 2 μl of 0.1 M DTT and 1 μl of the RnasaOUT ™ Recombinant Ribonuclease Inhibitor (40 units / μl, Invitrogen) were added. The contents of the tube were mixed gently and incubated at 42 ° C for 2 minutes, then 1 µl (200 units) of the SuperScript II ™ enzyme was added and mixed gently by pipetting. The mixture was incubated at 42 ° C for 50 minutes and then quenched by heating at 70 ° C for 15 minutes. To extract the complementary RNA from the cDNA, 1 µl (2 units) of E. coli RNase H (Invitrogen) was added and the reaction mixture was incubated at 37 ° C for 20 minutes.

For SuperScript III: 1 μl of Oligo primer (dT) 20 (50 μM, Invitrogen), 2 μg of total human RNA, 1 μl of the 10 mM dNTP mixture (10 mM each of the dATP, dGTP, dCTP) was combined and dTTP at neutral pH) and sterile distilled water at a final volume of 10 μl, in a 1.5 ml Eppendorf tube and heated at 65 ° C for 5 minutes and then cooled with ice. For each reverse transcriptase reaction, a cDNA synthesis mixture was prepared as follows: 2 μl of 10 × RT buffer solution, 4 μl of 25 mM MgCl 2, 2 μl of 0.1M DTT, 1 μl of RNasaOUT ™ (40 U / μl) and 1 μl of SuperScript III ™ enzyme reverse transcriptase in a separate tube and then 10 μl of this mixture was added to the tube containing the RNA / primer mixture. The contents of the tube were mixed gently, collected by brief centrifugation and incubated at 50 ° C for 50 minutes. The reaction was terminated by incubation at 80 ° C for 5 minutes and the reaction mixture was then cooled with ice and collected by brief centrifugation. To extract the complementary RNA from the cDNA, 1 µl (2 units) of E. coli RNase H (Invitrogen) was added and the reaction mixture was incubated at 37 ° C for 20 minutes.

The final reaction mixture of 21 μl was diluted by the addition of 179 μl of sterile water to provide a total volume of 200 μl. RNA samples were combined in groups such that each group contained five different samples of cDNA. 5 µl of each cDNA group was used as a model for PCR in a final reaction volume of 50 µl and this consisted of 1 µl of each cDNA sample in that group. This represented approximately 20 ng of each individual cDNA model.

b) cDNA libraries

The human cDNA libraries (in lambda (λ) bacteriophage vectors) were purchased from Stratagene, Clontech or Invitrogen or prepared at the Serono Pharmaceutical Research Institute in λ ZAP, λ GTI10, λ GT11, or TriplEx2 vectors in accordance with the protocol from the manufacturer (Stratagene, Clontech and Invitrogen). The bacteriophage λ DNA was prepared from small-scale cultures of the E. coli host strain infested by using the Wizard Lambda Preps DNA purification system. in accordance with the manufacturer's instructions (Promega, Corporation, Madison Wis.).

c) Gene specific cloning primers for PCR

Two pairs of PCR primers with a length of between 18 and 25 bases were designed to amplify sequences 3 and 4 of the sCSF3R exons by using the First Designer Software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722-2045, USA). PCR primers were improved to have a Tm near 55 ± 10 ° C and a GC content of 40-60%. Primers were selected that had high selectivity for the white sequence (sCSF3R) with little or no specific priming. The primers were designed to form two nested pairs so that the nested sCSF3R-F1 / sCSF3R-R1 primers were positioned slightly inside the sCSF3R-F1 / sCSF3R-R1 primers.

d) PCR amplification of the human cDNA models of the SCSF3R.

The sCSF3R-F1 / sCSF3R-R1 gene-specific cloning primers (Table 3, FIG. 1) were designed to amplify a 554 bp cDNA fragment within exons 3 and 4 of sCSF3R. The pair of sCSF3R-F1 / sCSF3R-R1 primers was used in PCR1 on the panel of cDNA library models and groups of cDNA models of individual tissues. This PCR1 was performed in a final volume of 50 μl containing 1 × AmpliTaq ™ buffer solution, 200 μM dNTPs, 50 pmoles of each cloning primer, 2.5 units of AmpliTaq ™ (Applied Biosystems) and approximately 20 ng of the cDNA library or 100 ng of the grouped cDNA model by using an MJ DNA search engine, programmed as follows: 94 ° C, 2 minutes; 40 cycles of 94 ° C, 1 minute, 57 ° C, 1 minute and 72 ° C, 1 minute; followed by 1 cycle at 72 ° C for 7 minutes and a retention cycle at 4 ° C, 30 μl of each PCR1 amplification product was visualized on a 0.8% agarose gel in 1 × TAE buffer solution (Invitrogen ) but no products were observed.

Each product of PCR1 was then used as a model for PCR2 through the use of nested sCSF3R-F1 / sCSF3R-R1 amplification primers (Table 3, FIG. 1-4), designed to amplify the 500 bp cDNA fragment within of the product sCSF3R-F1 / sCSF3R-R1. PCR2 was performed in a final volume of 50 μl containing a 1 × AmpliTaq ™ buffer solution, 200 μM dNTPs, 50 pmoles of each cloning primer, 2.5 units of AmpliTaq ™ (Applied Biosystems) and 1 μl of the product of PCR1 by using an MJ DNA search engine, programmed as follows: 94 ° C, 2 minutes; 40 cycles of 94 ° C, 1 minute, 61 ° C, 1 minute and 72 ° C, 1 minute; followed by 1 cycle at 72 ° C for 7 minutes and a retention cycle at 4 ° C.

30 µl of each PCR2 amplification product was visualized on a 0.8% agarose gel in 1 × TAE buffer solution (Invitrogen). The products of approximately the expected molecular weight were purified from the gel by using the Qiagen MinElute (Qiagen) DNA purification system, eluted in 10 μl of EB buffer solution (10 mM Tris.Cl, pH 8.5) and they were directly subcloned.

e) Subcloning of PCR products

The PCR products were subcloned into the topoisomerase I modified cloning vector (pCR4-TOPO) by using the reverse transcriptase cloning kit of Invitrogen Corporation under the conditions specified by the manufacturer. Briefly, 4 μl of the purified gel gel product was incubated for 15 minutes at room temperature with 1 μl of the TOPO vector and 1 μl of saline. The reaction mixture was then transformed into E. coli strain TOP10 (Invitrogen) as follows: a 50 μl aliquot of TOP10 One Shot cells was thawed on ice and 2 μl of the TOPO reaction was added. The mixture was incubated for 15 minutes on ice and then subjected to thermal shock by incubation at 42 ° C for exactly 30 seconds. The samples were returned to ice and 250 µl of a tempered SOC medium (room temperature) was added. The samples were incubated with shaking (220 rpm) for 1 hour at 37 ° C. The transformation mixture was then placed in broth plates L (LB) containing ampicillin (100 μg / ml) and incubated overnight at 37 ° C.

f) Colony PCR

The colonies were inoculated in 50 µl of sterile water by using a sterile toothpick. A 10 μl aliquot of the inoculum was then subjected to PCR in a total reaction volume of 20 μl containing 1 × AmpliTaq ™ buffer solution, 200 μM dNTPs, 20 pmoles of T7 primer, 20 pmoles of T3 primer and 1 unit of AmpliTaq (Applied Biosystems) by using an MJ DNA search engine. The cycle conditions were as follows: 94 ° C., 2 minutes; 30 cycles of 94 ° C., 30 seconds, 48 ° C., 30 seconds and 72 ° C. for 1 minute. Samples were kept at 4 ° C (retention cycle) before further analysis.

The PCR reaction products on 1% agarose gels in 1 × TAE buffer solution were analyzed. Colonies that provided PCR products of approximately the expected molecular weight (500 bp + 105 bp due to the multiple cloning site (MCS)) were grown overnight at 37 ° C in an L-broth (LB) of 5 ml containing ampicillin (100 μg / ml), with agitation at 220 rpm.

f) Preparation and sequencing of plasmid DNA

Plasmid DNA Miniprep was prepared from the 5 ml culture by using the Biorobot 8000 robotic system (Qiagen) or the Wizard Plus SV Minipreps kit (Promega cat. No. 1460) in accordance with the manufacturer's instructions. Plasmid DNA was eluted in 80 µl of sterile water. The DNA concentration was measured by using the Spectramax 190 (Molecular Devices) photometer. Plasmid DNA (200-500 ng) was subjected to DNA sequencing with primers T7 and T3 (Table 3) by using the BigDye terminator system (Applied Biosystems cat. No. 4390246) in accordance with the manufacturer's instructions. The sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleaning plates (Millipore cat. No. LSKS09624) and then analyzed on an Applied Biosystems 3700 sequencer.

Sequence analysis identified a clone, amplified from a group (PS3) containing cDNA derived from the mixed RA synovial membrane, mixed osteoarthritis cells and pulmonary fibroblasts containing the sCSF3R sequence. In the cloned sequence, 27 bp was missing at the 5 ′ end and 40 bp was missing at the 3 ′ end due to the position of the nested primers used for the PCR. The sequence of the cloned cDNA fragment is shown in FIG. 2. The cloned PCR product is found in the plasmid pCR4-TOPO-sCSF3RF1 / R1.

Construction of mammalian cell expression vectors for the sCSF3R The plasmid nested pCR4-TOPO-sCSF3RF1 / R1 was used as a PCR model to generate the expression clones pEAK12d and pDEST12.2 containing the sCSF3R ORF sequence with a 3 ′ sequence that encode the 6HIS marker by using the Gateway ™ cloning methodology (Invitrogen).

a) Generation of the open reading frame (ORF) of the compatible sCSF3R Gateway merged with an incoming frame 6HIS marker sequence.

The first stage of the Gateway cloning process involves a four-stage PCR reaction that generates the ORF of the sCSF3R flanked at the 5 ′ end by a recombination site attB1 and a Kozak sequence and flaked at the 3 ′ end by a sequence qu and encodes an incoming frame histidine 6 (6HIS) marker, an end codon and the attB2 recombination site (Gateway compatible cDNA). The missing bases of the planned CDs were added with the sense primers sCSF3R-EX1, sCSF3R-EX3 and the antisense primers sCSF3R-EX2, sCSF3R-EX4.

The first PCR reaction (to add 14 bases at the 5 ′ end and 18 bases at the 3 ′ end) contains respectively (in a final volume of 50 μl): 1 μl (30 ng) of pCR4-TOPO-sCSF3RF1 / R1 plasmid, 1.5 μl of dNTPs (10 mM), 10 μl of 10 × Pfx polymerase buffer solution, 1 μl of MgSO4 (50 mM), 0.5 μl of each of the gene specific primers (100 μM) (sCSF3R-EX1 and sCSF3R-EX2) and 0.5 μl of Platinum Pfx DNA polymerase (Invitrogen). The PCR reaction was performed by using an initial denaturing step of 95 ° C for 2 minutes, followed by 20 cycles of 94 ° C for 15 seconds; 55 ° C. for 30 seconds and 68 ° C. for 2 minutes and a retention cycle of 4 ° C.

A 10 μl aliquot was observed on the 0.8% agarose gel in a 1 × TAE buffer solution (Invitrogen) to verify that the product had the expected molecular weight (500 ± 32 = 532 bp). The remaining 40 μl were loaded onto the 0.8% agarose gel in a 1 × TAE buffer gel and the band was purified using the Wizard PCR Preps DNA purification system (Promega) and recovered in 50 μl of sterile water in accordance with the manufacturer's instructions. The second PCR reaction (to add the remaining nucleotides at both ends) contains respectively (in a final volume of 50 μl): 4 μl (4 ng) of the purified PCR product1, 1.5 μl of dNTPs (10 mM) , 10 μl of 10 × Pfx polymerase buffer solution, 1 μl of MgSO4 (50 mM), 0.5 μl of each of the gene-specific primers (100 μM) (sCSF3R-EX3 and sCSF3R-EX4) and 0, 5 μl of Platinum Pfx DNA polymerase (Invitrogen). The PCR reaction was performed by using an initial denaturing step of 95 ° C for 2 minutes, followed by 20 cycles of 94 ° C for 15 seconds; 55 ° C. for 30 seconds and 68 ° C. for 2 minutes and a 4 ° C retention cycle. A 10 μl aliquot was observed on the 0.8% agarose gel in a 1 × TAE buffer solution ( Invitrogen) to verify that the product had the expected molecular weight. The remaining 40 μl was loaded onto the 0.8% agarose gel in a 1 × TAE buffer gel and the band was purified using the Wizard SV Gel and the PCR cleaning system (Promega) and recovered in 40 μl of sterile water in accordance with the manufacturer's instructions.

The third PCR reaction (in a final volume of 50 μl) contains respectively: 4 μl of the purified PCR2 product, 1.5 μl of dNTPs (10 mM), 10 μl of 10 × Pfx polymerase buffer solution, 1 μl MgSO4 (50 mM), 0.5 μl of each of the gene-specific primers (100 μM) (sCSF3R-EX5 and sCSF3R-EX6) and 0.5 μl of Platinum Pfx DNA polymerase (Invitrogen). The PCR reaction was performed by using an initial denaturing step of 95 ° C for 2 minutes, followed by 12 cycles of 94 ° C for 15 seconds; 55 ° C. for 30 seconds and 68 ° C. for 2 minutes and a 4 ° C retention cycle. The amplification product was directly purified by using the Wizard PCR Preps DNA purification system (Promega) and recovered in 50 μl of sterile water in accordance with the manufacturer's instructions.

The fourth PCR reaction (in a final volume of 50 μl) contains 10 μl of the purified PCR3 product, 1.5 μl of dNTPs (10 mM), 10 μl of 10 × Pfx polymerase buffer solution, 1 μl of MgSO4 (50 mM), 0.5 μl of each of the Gateway conversion primers (100 μM) (sense GCP and antisense GCP) and 0.5 μl of Platinum Pfx DNA polymerase. The conditions for the second PCR reaction were as follows: 95 ° C for 1 minute; 4 cycles of 94 ° C, 15 seconds; 50 ° C, 30 seconds and 68 ° C for 2 minutes; 25 cycles of 94 ° C, 15 seconds; 55 ° C, 30 seconds and 68 ° C, 2 minutes; followed by a 4 ° C cycle. A 10 μl aliquot was observed on the 0.8% agarose gel in a 1 × TAE buffer solution (Invitrogen) to verify that the product had the expected molecular weight (567 ± 70 = 637 bp). The remaining 40 μl were loaded onto the 0.8% agarose gel in a 1 × TAE buffer gel and the band was purified using the Wizard PCR Preps DNA purification system (Promega) and recovered in 50 μl of sterile water in accordance with the manufacturer's instructions.

b) Subcloning the open reading frame (ORF) of the Gateway compatible sCSF3R into the Gateway input vector pDONR221 and the expression vectors pEAK12d and pDEST12.2.

The second stage of the Gateway cloning process involves subcloning the modified PCR Gateway product into the Gateway pDONR221 (Invitrogen) input vector as follows: 5 µl of the purified PCR4 product was incubated with 1.5 µl of the pDONR221 vector (0.1 μg / μl), 2 μl of BP buffer solution and 1.5 μl of a mixture of the BP clonase enzyme (Invitrogen) in a final volume of 10 μl at room temperature for 1 hour. The reaction was stopped by the addition of 1 µl of proteinase K (2 µg / µl) and incubated at 37 ° C for an additional 10 minutes. An aliquot of this reaction (1 μl) was used to transform E. coli DH10B cells by electro

poration as follows: a 25 μl aliquot of DH10B electrocompetent cells (Invitrogen) was thawed on ice and 1 μl of the BP reaction mixture was added. The mixture was transferred to a cold 0.1 cm electroporation cuvette and the cells were electroporized by using a BioRad Gene-Pulser ™ apparatus in accordance with the manufacturer's recommended protocol. Immediately after electroporation, a SOC medium (0.5 ml) was added which had been preheated to room temperature. The mixture was transferred to a 15 ml pressure cap tube and incubated with stirring (220 rpm) for 1 hour at 37 ° C. The aliquots of the transformation mixture (10 μl and 50 μl) were then placed on plates of L-broth (LB) containing kanamycin (40 μg / ml) and incubated overnight at 37 ° C.

The miniprep plasmid DNA was prepared from a 5 ml culture of 8 of the resulting colonies by using a Qiaprep BioRobot 8000 system (Qiagen). Plasmid DNA (150-200 ng) was subjected to DNA sequencing with primers 21M13 and M13Rev by using the BigDye terminator system (Applied Biosystems cat. No. 4336919) in accordance with the manufacturer's instructions. The primer sequences are shown in the Table

3. Sequencing reactions were purified using Montage SEQ 96 cleaning plates (Millipore cat. No. LSKS09624) and then analyzed on an Applied Biosystems 3700 sequencer.

The plasmid eluate (2 μl or approximately 150 ng) from one of the clones containing the correct sequence (pENTR_sCSF3R-6HIS) was then used in a recombination reaction containing 1.5 μl of the pE-AK12d vector or vector (0 , 1 μg / μl), 2 μl of LR buffer solution and 1.5 μl of LR clonase (Invitrogen) in a final volume of 10 μl. The mixture was incubated at room temperature for 1 hour, stopped by the addition of proteinase K (2 μg) and incubated at 37 ° C for an additional 10 minutes. An aliquot of this reaction (1 ul) was used to transform E. coli DH10B cells by electroporation as follows: a 25 μl aliquot of DH10B electrocompetent cells (Invitrogen) was thawed on ice and 1 μl of the LR reaction mixture. The mixture was transferred to a cold 0.1 cm electroporation cuvette and the cells were electroporized by using a BioRad Gene-Pulser ™ apparatus in accordance with the manufacturer's recommended protocol. Immediately after electroporation, a SOC medium (0.5 ml) was added which had been preheated to room temperature. The mixture was transferred to a 15 ml pressure cap tube and incubated with stirring (220 rpm) for 1 hour at 37 ° C. The aliquots of the transformation mixture (10 μl and 50 μl) were then placed on plates of L-broth (LB) containing ampicillin (100 μg / ml) and incubated overnight at 37 ° C.

The miniprep plasmid DNA was prepared from the 5 ml cultures of 6 of the resulting colonies subcloned into each vector by using a Qiaprep BioRobot 8000 system (Qiagen). Plasmid DNA (200-500 ng) in vector pEAK12d was subjected to DNA sequencing with primers pEAK12F and pEAK12R as described above. Plasmid DNA (200-500 ng) in vector pDEST12.2 was subjected to DNA sequencing with primers 21M13 and M13Rev as described above. The primer sequences are shown in the Table

3.

The maxi-prep DNA purified by CsCl gradient was prepared from a 500 ml culture of the verified sequence clone (pEAK12d_sCSF3R-6HIS) by using the method described by Sambrook J. et al., 1989 (in Molecular Cloning , a Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press). Plasmid DNA was resuspended at a concentration of 1 μg / μl in sterile water (or 10 mM Tris-HCl pH 8.5) and stored at −20 ° C.

Endotoxin-free maxi-prep DNA was prepared from a 500 ml culture of the verified sequence clone (pDEST12.2_sCSF3R-6HIS) by using the EndoFree Plasmid Mega (Qiagen) kit in accordance with the manufacturer's instructions. The purified plasmid DNA was resuspended in an endotoxin-free TE buffer solution at a final concentration of at least 3 μg / μl and stored at −20 ° C.

TABLE 3: SCSF3R cloning and sequencing primers

Primer Sequence (5′-3 ′)

SCSF3R-F1 ATGGCAAGGCTGGGAAACTG

SCSF3R-R1 TTTAGTAGAGGCGGGGTTTCG

SCSF3R-F1 nested ACTTGGGCTGCCCTGATCATCC

SCSF3R-R1 nested TGGCCAGGCTGATCATGAACTC

SCSF3R-EX1 GAAACTGCAGCCTGACTTGGGCTGCCCTGATCA

SCSF3R-EX2 GAGGCGGGGTTTCGCCTTGCTGGCCAGGCTGATCAT

GAAC

SCSF3R-EX3 ATGGCAAGGCTGGGAAACTGCAGCCTGACTT

SCSF3R-EX4 ATTTTTTTGTATTTTTAGTAGAGGCGGGGTTTCGCC

TT

SCSF3R-EX5 GCAGGCTTC GCCACC ATGGCAAGGCTGGGAAACTG

SCSF3R-EX6 TGATGGTGATGGTG ATTTTTTTGTATTTTTAGTAGA

GGC

GCP sense

GGGGACAAGTTTGTACAAAAAAGCAGGCTTC GCCA

DC

Antisense GCP

GGGGACCACTTTGTACAAGAAAGCTGGGTT TCA ATG

GTGATGGTGATGGTG

pEAK12F

GCCAGCTTGGCACTTGATGT

pEAK12R

GATGGAGGTGGACGTGTCAG

21M13

TGTAAAACGACGGCCAGT

M13REV

CAGGAAACAGCTATGACC

T7

TAATACGACTCACTATAGG

T3

ATTAACCCTCACTAAAGG

Underlined Sequence = Bold Kozak Sequence = Finish Code Italic Sequence = His Marker

Example 2 - Expression of functional genomics in mammalian cells and purification of the His-labeled cloned plasmid pEAK12d_sCSF3R-6HIS

293 human embryonic kidney cells expressing the nuclear antigen of the Epstein-Barr virus (HEK293-EBNA, Invitrogen) were suspended in a serum-free medium Ex-cell VPRO (stock in broth, maintenance medium, JRH). The cells were inoculated in a medium at 1 × 10 6 cells / ml in 250 ml FEME (DMEM / Ham's F

12 1: 1 19 mM HEPES, 5 g / L Glucose, 7.5 mM L-Glutamine, 4 ml / L ITS-X) (all Invitrogen-Life Technologies) supplemented with 1% FCS. For the transfection mixture, 500 μg of DNA (pEAK12d_sCSF3R-6HIS) was diluted plus 10 μg of the reporter gene DNA in 50 ml FEME 1% FCS. Then 1 ml of PEI (1 mg / l Polysciences, USA) was added. This mixture was incubated for 10 minutes at room temperature. After 10 minutes the transfection mixture was added to the cells and the culture was incubated at 37 ° C in the incubator for 90 minutes. Finally, the volume was completed with the 200 ml of 1% FCS FEME containing 2.5 ml of Pen-Strep to prevent contamination due to DNA instability. Confirmation of positive transfection was performed by qualitative fluorescence examination on day 6 (Axiovert 10 Zeiss). On day 6 (harvest day), the supernatant (500 ml) was centrifuged (4 ° C, 400 g) and placed in a container that had a unique identifier.

An aliquot (500 μl) was retained for quality control (QC) of the 6His labeled protein (internal bioprocess quality control).

Purification process

The sample of the 500 ml culture medium containing the recombinant protein with a 6His C-terminal marker was diluted with a frozen buffer solution A (50 mM NaH 2 PO 4; 600 mM NaCl; 8.7% glycerol (w / v), pH 7.5) at a final volume of 1000 ml. The sample was filtered through a 0.22 μm sterile filter (Millipore, 500 ml filter unit) and kept at 4 ° C in a sterile 1 liter square bottle (Nalgene).

Purification was performed at 4 ° C in a VISION work terminal (Applied Biosystems) connected to an automatic sample loader (Labomatic). The purification procedure was composed of two sequential stages, metal affinity chromatography on a Poros 20 MC (Applied Biosystems) column loaded with Ni ions (10 × 50 mm, 3.93 ml), followed by a solution exchange buffer on a Sephadex G-25 gel filtration column (Amersham Pharmacia) (1.0 × 15 cm).

For the first chromatographic stage the metal affinity column was regenerated with 30 column volumes of EDTA solution (100 mM EDTA; 1 M NaCl; pH 8.0), recharged with Ni ions by washing with 15 column volumes of a 100 mM NiSO 4 solution, washed with 10 column volumes of buffer solution A, followed by 7 column volumes of buffer solution B (50 mM NaH 2 PO 4; 600 mM NaCl; 8.7 glycerol % (w / v), 400 mM; imidazole, pH 7.5) and finally equilibrated with 15 column volumes of buffer solution A containing 15 mM imidazole. The sample was transferred, by means of the Labomatic sample loader, to a 200 ml sample handle and then loaded onto an affinity column of the Ni metal at a flow rate of 20 ml / min. The loading procedure was repeated 5 times to transfer the entire sample (1000 ml) to the Ni column. The column was then washed with 12 column volumes of buffer solution A followed by 28 column volumes of buffer solution A containing 20 mM imidazole. During the 20 mM imidazole wash the slightly bound contaminating proteins were eluted from the column. The recombinant His-labeled protein was finally eluted with 10 column volumes of buffer solution B at a flow rate of 2 ml / min and the eluted protein was collected in a fraction of 2.7 ml.

For the second chromatography step, the Sephadex G-25 gel filtration column was regenerated with 2 ml of buffer solution D (1,137 M NaCl; 2.7 mM KCl; 1.5 mM KH 2 PO 4; Na 2 HPO 4 8 mM; pH 7.2), and then equilibrated with 4 column volumes of buffer solution C (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH 2 PO 4; 8 mM Na 2 HPO 4; 20% glycerol (w / v); pH 7.4). The eluted peak fraction of the Ni column was automatic, by means of the sample loader integrated on the VISION, loaded on the Sephadex G-25 column and the protein was eluted with buffer solution C at a flow rate of 2 ml / min. . The desalinated sample was recovered in a fraction of 2.7 ml. The fraction was filtered through a sterile 0.22 μm centrifuge filter (Millipore), separated into aliquots, frozen and stored at −80 ° C. An aliquot of the sample was analyzed on SDS-PAGE (4 -12% NuPAGE gel; Novex) by Coomassie blue staining and immunoblotting (Western blot) with anti-His antibodies.

Coomassie blue staining

The NuPAGE gel was stained in a 0.1% Coomassie R250 blue staining solution (30% methanol, 10% acetic acid) at room temperature for 1 hour and then stained in 20% methanol, acetic acid at 7.5% until the bottom was clean and the protein bands clearly visible.

Immunoblot (Western Blot)

Following electrophoresis, the proteins were electrotransferred from the gel to a nitrocellulose membrane at 290 mA for 1 hour at 4 ° C. The membrane was blocked with 5% milk powder in buffer solution E (137 mM NaCl; KCl 2 , 7 mM; 1.5 mM KH 2 PO 4; 8 mM Na 2 HPO 4; 0.1% Tween 20, pH 7.4) for 1 hour at room temperature and then incubated with a mixture of 2 anti antibodies - Polyclonal rabbit rabbits (G-18 and H-15, 0.2 ug / ml each; Santa Cruz) in 2.5% milk powder in buffer solution E overnight at 4 ° C. After After 1 hour of incubation at room temperature, the membrane was washed with buffer solution E (3x10 min.) and then incubated with a secondary anti-rabbit antibody conjugated with HRP (DAKO, HRP 0399) diluted 1/3000 in the buffer solution E containing 2.5% milk powder for 2 hours at room temperature. After washing with buffer solution E (3x10 minutes), the membrane was developed with the ECL kit (Amersham) for 1 minute. The membrane was then exposed to hyper film (Amersham), the film was developed and the immunoblot image (Western blot) was analyzed visually.

Protein test

Protein concentration was determined by using the BCA protein assay kit (Pierce) with bovine serum albumin as standard. The product was 184 μg of purified sCSF3R-6HIS.

SEQUENCE LIST

<110> Applied Research Systems ARS Holding N.V.

<120> CSF3R polypeptides and their uses

<130> w01130

<150> EP 06116704.5

<151> 2006-07-06

<150> US 60 / 832,177

<151> 2006-07-19

<160> 30

<170> PatentIn version 3.3

<210> 1

<211> 588

<212> DNA

<213> homo sapiens

<400> 1 atggcaaggc tgggaaactg cagcctgact tgggctgccc tgatcatcct gctgctcccc 60 ggaagtctgg aggagtgcgg gcacatcagt gtctcagccc ccatcgtcca cctgggggat 120 cccatcacag cctcctgcat catcaagcag aactgcagcc atctggaccc ggagccacag 180 attctgtgga gactgggagc agagcttcag cccgggggca ggcagcagcg tctgtctgat 240 gggacccagg aatctatcat caccctgccc cacctcaacc acactcaggc ctttctctcc 300 actggggcaa tgctgcctga cagcctgcag atcctggacc aggttgagct gcgcgcaggc 360 tgtaagtcct tccagccatc caactactct gcctccaaca ccctcctgcc aatactaata 420 agaatattac cagccgggca cgttggctca cgcctgtatt cccagcactt tgggaggccg 480 aggcaggcgg atcacctgag gtcaggagtt catgatcagc ctggccagca aggcgaaacc 540 ccgaactaccat

<210> 2

<211> 189

<212> PRT

<213> homo sapiens

<400> 2 Met Wing Arg Leu Gly Asn Cys Ser Leu Thr Trp Wing Wing Leu Ile Ile 15 10 15 Leu Leu Leu Pro Gly Ser Leu Glu Glu Cys Gly His Ile Ser Val Ser 20 25 30 Pro Wing Ile Val His Leu Gly Asp Pro Ile Thr Ala Ser Cys Ile Ile 35 40 45 Lys Gln Asn Cys Ser His Leu Asp Pro Glu Pro Gln Ile Leu Trp Arg 50 55 60 Leu Gly Ala Glu Leu Gln Pro Gly Gly Arg Gln Gln Arg Leu Ser Asp 65 70 75 80

Gly Thr Gln Glu Ser Ile Ile Thr Leu Pro His Leu Asn His Thr Gln 85 90 95 Ala Phe Leu be cys cys Leu Asn Trp Gly Asn be Leu Gln Ile Leu 100 105 110 Asp Gln Val Glu Leu Arg Wing Gly Cys Lys Ser Phe Gln Pro Ser Asn 115 120 125 Tyr Ser Ala Ser Asn Thr Leu Leu Pro Ile Leu Ile Arg Ile Leu Pro 130 135 140 Ala Gly His Val Gly Ser Arg Leu Tyr Ser Gln His Phe Gly Arg Pro 145 150 155 160 Arg Gln Ala Asp His Leu Arg Ser Gly Val His Asp Gln Pro Gly Gln 165 170 175 Gln Gly Glu Thr Pro Pro Leu Leu Lys Ile Gln Lys Asn 180 185

<210> 3

<211> 513

<212> DNA

<213> homo sapiens

<400> 3 gagtgcgggc acatcagtgt ctcagccccc atcgtccacc tgggggatcc catcacagcc 60 tcctgcatca tcaagcagaa ctgcagccat ctggacccgg agccacagat tctgtggaga 120 ctgggagcag agcttcagcc cgggggcagg cagcagcgtc tgtctgatgg gacccaggaa 180 tctatcatca ccctgcccca cctcaaccac actcaggcct ttctctcctg ctgcctgaac 240 tggggcaaca gcctgcagat cctggaccag gttgagctgc gcgcaggctg taagtccttc 300 cagccatcca actactctgc ctccaacacc ctcctgccaa tactaataag aatattacca 360 gccgggcacg ttggctcacg cctgtattcc cagcactttg ggaggccgag gcaggcggat 420 cacctgaggt caggagttca tgatcagcct ggccagcaag gcgaaacccc gcctctacta 480 aaaatacaaa aaaatcacca tcaccatcac cat 513

<210> 4

<211> 165

<212> PRT

<213> homo sapiens

<400> 4 Glu Cys Gly His Ile Ser Val Ser Ala Pro Ile Val His Leu Gly Asp 15 10 15 Pro Ile Thr Ala Ser Cys Ile Ile Lys Gln Asn Cys Ser His Leu Asp 20 25 30

Pro Glu Pro Gln Ile Leu Trp Arg Leu Gly Ala Glu Leu Gln Pro Gly 3540 45 Gly Arg Gln Gln Arg Leu Be Asp Gly Thr Gln Glu Ser Ile Ile Thr 50 55 60 Leu Pro His Leu Asn His Thr Gln Wing Phe Leu Ser Cys Cys Leu Asn 65 70 75 80 Trp Gly Asn Ser Leu Gln Ile Leu Asp Gln Val Glu Leu Arg Wing Gly 8590 95 Cys Lys Be Phe Gln Pro Be Asn Tyr Be Wing Be Asn Thr Leu Leu 100 105 110 Pro Ile Leu Ile Arg Ile Leu Pro Wing Gly His Val Gly Ser Arg Leu 115 120 125 Tyr Ser Gln His Phe Gly Arg Pro Arg Gln Wing Asp His Leu Arg Ser 130 135 140 Gly Val His Asp Gln Pro Gly Gln Gln Gly Glu Thr Pro Pro Leu Leu 145 150 155 160 Lys Ile Gln Lys Asn 165

<210> 5

<211> 501

<212> DNA

<213> homo sapiens

<400> 5 acttgggctg ccctgatcat cctgctgctc cccggaagtc tggaggagtg cgggcacatc 60

agtgtctcag cccccatcgt ccacctgggg gatcccatca cagcctcctg catcatcaag 120

cagaactgca gccatctgga cccggagcca cagattctgt ggagactggg agcagagctt 180

cagcccgggg gcaggcagca gcgtctgtct gatgggaccc aggaatctat catcaccctg 240

ccccacctca accacactca ggcctttctc tcctgctgcc tgaactgggg caacagcctg 300

cagatcctgg accaggttga gctgcgcgca ggctgtaagt ccttccagcc atccaactac 360

tctgcctcca acaccctcct gccaatacta ataagaatat taccagccgg gcacgttggc 420

tcacgcctgt attcccagca ctttgggagg ccgaggcagg cggatcacct gaggtcagga 480

gttcatgatc agcctggcca g 501

<210> 6

<211> 167

<212> PRT

<213> homo sapiens

<400> 6

41

Thr Trp Wing Wing Leu Ile Ile Leu Leu Leu Pro Gly Ser Leu Glu Glu 15 10 15

Cys Gly His Ile Ser Val Ser Ala Pro Ile Val His Leu Gly Asp Pro 20 25 30 Ile Thr Ala Ser Cys Ile Ile Lys Gln Asn Cys Ser His Leu Asp Pro 35 40 45 Glu Pro Gln Ile Leu Trp Arg Leu Gly Ala Glu Leu Gln Pro Gly Gly 50 55 60 Arg Gln Gln Arg Leu Ser Asp Gly Thr Gln Glu Ser Ile Ile Thr Leu 65 70 75 80

Pro His Leu Asn His Thr Gln Ala Phe Leu Ser Cys Cys Leu Asn Trp 85 90 95 Gly Asn Ser Leu Gln Ile Leu Asp Gln Val Glu Leu Arg Ala Gly Cys 100 105 110 Lys Ser Phe Gln Pro Ser Asn Tyr Ser Ala Ser Asn Thr Leu Leu Pro 115 120 125

Ile Leu Ile Arg Ile Leu Pro Wing Gly His Val Gly Ser Arg Leu Tyr 130 135 140

Ser Gln His Phe Gly Arg Pro Arg Gln Ala Asp His Leu Arg Ser Gly 145 150 155 160

val His Asp Gln Pro Gly Gln 165

<210> 7

<211> 456

<212> DNA

<213> homo sapiens

<400> 7 gagtgcgggc acatcagtgt ctcagccccc atcgtccacc tgggggatcc catcacagcc

tcctgcatca tcaagcagaa ctgcagccat ctggacccgg agccacagat tctgtggaga

ctgggagcag agcttcagcc cgggggcagg cagcagcgtc tgtctgatgg gacccaggaa

tctatcatca ccctgcccca cctcaaccac actcaggcct ttctctcctg ctgcctgaac

tggggcaaca gcctgcagat cctggaccag gttgagctgc gcgcaggctg taagtccttc

cagccatcca actactctgc ctccaacacc ctcctgccaa tactaataag aatattacca

gccgggcacg ttggctcacg cctgtattcc cagcactttg ggaggccgag gcaggcggat

cacctgaggt caggagttca tgatcagcct ggccag

<210> 8

<211> 152

<212> PRT

<213> homo sapiens

<400> 8

120 180 240 300 360 420 456

Glu Cys Gly His Ile Ser Val Ser Ala Pro Ile Val His Leu Gly Asp 15 10 15 Pro Ile Thr Ala Ser Cys Ile Ile Lys Gln Asn Cys Ser His Leu Asp 20 25 30 Pro Glu Pro Gln Ile Leu Trp Arg Leu Gly Ala Glu Leu Gln Pro Gly 35 40 45 Gly Arg Gin Gln Arg Leu Ser Asp Gly Thr Gin Glu Ser Ile Ile Thr 50 55 60 Leu Pro His Leu Asn His Thr Gln Wing Phe Leu Ser Cys Cys Leu Asn 65 70 75 80 Trp Gly Asn Ser Leu Gin Ile Leu Asp Gln Val Glu Leu Arg Wing Gly 85 90 95 Cys Lys Be Phe Gln Pro Be Asn Tyr Be Wing Be Asn Thr Leu Leu 100 105 110 Pro Ile Leu Ile Arg Ile Leu Pro Wing Gly His Val Gly Ser Arg Leu 115 120 125 Tyr Ser Gln His Phe Gly Arg Pro Arg Gln Wing Asp His Leu Arg Ser 130 135 140 Gly Val His Asp Gln Pro Gly Gln 145 150

<210> 9

<211> 2933

<212> DNA

<213> homo sapiens

<400> 9 ctggactgca gctggtttca ggaacttctc ttgacgagaa gagagaccaa ggaggccaag 60

caggggctgg gccagaggtg ccaacatggg gaaactgagg ctcggctcgg aaaggtgaag 120

taacttgtcc aagatcacaa agctggtgaa catcaagttg gtgctatggc aaggctggga 180

aactgcagcc tgacttgggc tgccctgatc atcctgctgc tccccggaag tctggaggag 240

tgcgggcaca tcagtgtctc agcccccatc gtccacctgg gggatcccat cacagcctcc 300

tgcatcatca agcagaactg cagccatctg gacccggagc cacagattct gtggagactg 360

ggagcagagc ttcagcccgg gggcaggcag cagcgtctgt ctgatgggac ccaggaatct 420

atcatcaccc tgccccacct caaccacact caggcctttc tctcctgctg cctgaactgg 480 ggcaacagcc tgcagatcct ggaccaggtt gagctgcgcg caggctaccc tccagccata 540

ccccacaacc tctcctgcct catgaacctc acaaccagca gcctcatctg ccagtgggag 600 ccaggacctg agacccacct acccaccagc ttcactctga agagtttcaa gagccggggc 660 cccaagggga aactgtcaga ctccatcctg gactgcgtgc ccaaggacgg gcagagccac 720 cacgcaaaca tgctgcatcc cctgctgttg taccagaata tgggcatctg ggtgcaggca 780 gagaatgcgc tggggaccag catgtcccca caactgtgtc ttgatcccat ggatgttgtg 840 aaactggagc cccccatgct gcggaccatg gaccccagcc ctgaagcggc ccctccccag 900 gcaggctgcc tacagctgtg ctgggagcca tggcagccag gcctgcacat aaatcagaag 960 tgtgagctgc gccacaagcc gcagcgtgga gaagccagct gggcactggt gggccccctc 1020 cccttggagg cccttcagta tgagctctgc gggctcctcc cagccacggc ctacaccctg 1080 cagatacgct gcatccgctg gcccctgcct ggccactgga gcgactggag ccccagcctg 1140 gagctgagaa ctaccgaacg ggcccccact gtcagactgg acacatggtg gcggcagagg 1200 cagctggacc ccaggacagt gcagctgttc tggaagccag tgcccctgga ggaagacagc 1260 ggacggatcc aaggttatgt ggtttcttgg agaccctcag gccaggctgg ggccatcctg 1320 acaccacaga cccctctgca gctcagctgc accttccacc tgccttcaga agcccaggag 1380 gtggcccttg tg gcctataa ctcagccggg acctctcgcc ccaccccggt ggtcttctca 1440 gaaagcagag gcccagctct gaccagactc catgccatgg cccgagaccc tcacagcctc 1500 tgggtaggct gggagccccc caatccatgg cctcagggct atgtgattga gtggggcctg 1560 gcgcgagcaa ggccccccca tagcaacaag acctggagga tggaacagaa tgggagagcc 1620 tgctgaagga acggggtttc gaacatcagg ccctttcagc tctatgagat catcgtgact 1680 cccttgtacc aggacaccat gggaccctcc cagcatgtct atgcctactc tcaagaaatg 1740 atgccccaga gctccctccc gctgcatcta aagcacattg gcaagacctg ggcacagctg 1800 gagtgggtgc ctgagccccc tgagctgggg aagagccccc ttacccacta caccatcttc 1860 tggaccaacg ctcagaacca gtccttctcc gccatcctga atgcctcctc ccgtggcttt 1920 gtcctccatg gcctggagcc cgccagtctg tatcacatcc acctcatggc tgccagccag 1980 gctggggcca ccaacagtac agtcctcacc ctgatgacct tgaccccaga ggggtcggag 2040 ctacacatca tcctgggcct gttcggcctc ctgctgttgc tcacctgcct ctgtggaact 2100 gcctggctct gttgcagccc caacaggaag aatcccctct ggccaagtgt cccagaccca 2160 gctcacagca gcctgggctc ctgggtgccc acaatcatgg aggaggatgc cttccagctg 2220 gcacgcca cccggccttg cc catcaccaag ctcacagtgc tggaggagga tgaaaagaag 2280 gggagtccca ccggtgccct taacagctca gagacctgtg gcctccccac tctggtccag 2340 tccaggggga acctatgtgc cccaagagca gtttccaccc agccccaatc ccagtctggc 2400 accagcgatc aggtccttta tgggcagctg ctgggcagcc ccacaagccc agggccaggg 2460 cactatctcc gctgtgactc cactcagccc ctcttggcgg gcctcacccc cagccccaag 2520 tcctatgaga acctctggtt cccttgggga ccaggccagc ccctggtaac cccagcccca 2580 agccaggagg acgactgtgt ctttgggcca ctgctcaact tccccctcct gcaggggatc 2640

cgggtccatg ggatggaggc gctggggagc ttctagggct tcctggggtt cccttcttgg 2700

gcctgccttt taaaggcctg agctagctgg agaagagggg agggtccata agcccatgac 2760

taaaaactac cccagcccag gctctcacca tctccagtca ccagcatctc cctctcctcc 2820

caatctccat aggctgggcc tcccaggcga tctgcatact ttaaggacca gatcatgctc 2880

catccagccc cacccaatgg ccttttgtgc ttgtttccta taacttcagt att 2933

<210> 10

<211> 836

<212> PRT

<213> homo sapiens

<400> 10 Met Wing Arg Leu Gly Asn Cys Ser Leu Thr Trp Wing Wing Leu Ile Ile 15 10 15

Leu Leu Leu Pro Gly be Leu Glu Glu cys Gly His Ile be val ser

20 25 30

Ala Pro Ile val His Leu Gly Asp Pro Ile Thr Ala Ser Cys Ile Ile 35 40 45

Lys Gln Asn cys Ser HiS Leu Asp Pro Glu Pro Gln Ile Leu Trp Arg 50 55 60

Leu Gly Ala Glu Leu Gln Pro Gly Gly Arg Gln Gln Arg Leu Ser Asp

65 70 75 80

Gly Thr Gln Glu Ser Ile Ile Thr Leu Pro His Leu Asn His Thr Gln 85 90 95 Ala Phe Leu Ser Cys Cys Leu Asn Trp Gly Asn Ser Leu Gin Ile Leu 100 105 110

Asp Gln val Glu Leu Arg Wing Gly Tyr Pro Pro Wing Ile Pro His Asn 115 120 125 Leu Ser Cys Leu Met Asn Leu Thr Thr Ser Ser Leu Ile Cys Gln Trp 130 135 140

Glu Pro Gly Pro Glu Thr His Leu Pro Thr Ser Phe Thr Leu Lys Ser

145 150 155 160

Phe Lys Ser Arg Gly Asn Cys Gln Thr Gln Gly Asp Ser Ile Leu Asp 165 170 175

Cys Val Pro Lys Asp Gly Gln Ser His Cys Cys Ile Pro Arg Lys His 180 185 190 Leu Leu Leu Tyr Gln Asn Met Gly Ile Trp Val Gln Wing Glu Asn Wing 195 200 205 Leu Gly Thr Ser Met Ser Pro Gln Leu Cys Leu Asp Pro Met Asp Val 210 215 220

Val Lys Leu Glu Pro Pro Met Leu Arg Thr Met Asp Pro Ser Pro Glu 225 230 235 240 Ala Pro Pro Gln Wing Gly Cys Leu Gln Leu Cys Trp Glu Pro Trp

245 250 255 Gln Pro Gly Leu His Ile Asn Gln Lys Cys Glu Leu Arg His Lys Pro 260 265 270 Gln Arg Gly Glu Ala Ser Trp Ala Leu Val Gly Pro Leu Pro Leu Glu 275 280 285 Ala Leu Gln Tyr Glu Leu Cys Gly Leu Leu Pro Wing Thr Wing Tyr Thr 290 295 300

Leu Gln Ile Arg Cys Ile Arg Trp Pro Leu Pro Gly His Trp Ser Asp 305 310 315 320 Trp Ser Pro Ser Leu Glu Leu Arg Thr Thr Glu Arg Ala Pro Thr Val

325 330 335 Arg Leu Asp Thr Trp Trp Arg Gin Arg Gln Leu Asp Pro Arg Thr Val 340 345 350 Gln Leu Phe Trp Lys Pro Val Pro Leu Glu Glu Asp Ser Gly Arg Ile 355 360 365

Gln Gly Tyr Val Val Ser Trp Arg Pro Ser Gly Gln Wing Gly Wing Ile 370 375 380

Leu Pro Leu Cys Asn Thr Thr Glu Leu Ser Cys Thr Phe His Leu Pro 385 390 395 400 Ser Glu Ala Gln Glu Val Ala Leu Val Ala Tyr Asn Ser Ala Gly Thr

405 410 415 Ser Arg Pro Thr Pro Val Val Phe Ser Glu Ser Arg Gly Pro Ala Leu 420 425 430 Thr Arg Leu His Met Met Wing Arg Asp Pro His Ser Leu Trp Val Gly 435 440 445 Trp Glu Pro Pro Asn Pro Trp Pro Gln Gly Tyr Val Ile Glu Trp Gly

450 455 460 Leu Gly Pro Pro Be Wing Be Asn Be Asn Lys Thr Trp Arg Met Glu 465 470 475 480 Gln Asn Gly Arg Wing Thr Gly Phe Leu Leu Lys Glu Asn Ile Arg Pro

485 490 495 Phe Gln Leu Tyr Glu Ile Ile Val Thr Pro Leu Tyr Gln Asp Thr Met 500 505 510

Gly Pro Ser Gln His Val Tyr Ala Tyr Ser Gln Glu Met Ala Pro Ser 515 520 525

His Wing Pro Glu Leu His Leu Lys His Ile Gly Lys Thr Trp Wing Gln 530 535 540 Leu Glu Trp Val Pro Glu Pro Pro Glu Leu Gly Lys Ser Pro Leu Thr 545 550 555 560

His Tyr Thr Ile Phe Trp Thr Asn Wing Gln Asn Gln Ser Phe Ser Wing 565 570 575

Ile Leu Asn Ala Be Being Arg Gly Phe Val Leu His Gly Leu Glu Pro 580 585 590

Ala Ser Leu Tyr His Ile His Leu Met Ala Ala Ser Gln Ala Gly Ala 595 600 605 Thr Asn Ser Thr Val Leu Thr Leu Met Thr Leu Thr Pro Glu Gly Ser 610 615 620

Glu Leu His Ile Ile Leu Gly Leu Phe Gly Leu Leu Leu Leu Leu Thr 625 630 635 640 Cys Leu Cys Gly Thr Wing Trp Leu Cys Cys Ser Pro Asn Arg Lys Asn 645 650 655

Pro Leu Trp Pro Ser Val Pro Asp Pro Ala His Ser Ser Leu Gly Ser 660 665 670 Trp Val Pro Thr Ile Met Glu Glu Asp Ala Phe Gln Leu Pro Gly Leu 675 680 685 Gly Thr Pro Pro Ile Thr Lys Leu Thr Val Leu Glu Glu Asp Glu Lys 690 695 700

Lys Pro Val Pro Trp Glu Ser His Asn Ser Ser Glu Thr Cys Gly Leu 705 710 715 720 Pro Thr Leu val Gln Thr Tyr val Leu Gln Gly Asp Pro Arg Ala val 725 730 735 Ser Thr Gln Pro Gln Ser Gln Ser Gly Thr Ser Asp Gln val Leu Tyr 740 745 750 Gly Gln Leu Leu Gly Ser Pro Thr Ser Pro Gly Pro Gly His Tyr Leu 755 760 765 Arg Cys Asp Ser Thr Gln Pro Leu Leu Ala Gly Leu Thr Pro Ser Pro 770 775 780 Lys Ser Tyr Glu Asn Leu Trp Phe Gln Ala Ser Pro Leu Gly Thr Leu 785 790 795 800 Val Thr Pro Ala Pro Ser Gln Glu Asp Asp Cys Val Phe Gly Pro Leu 805 810 815 Leu Asn Phe Pro Leu Leu Gln Gly Ile Arg Val His Gly Met Glu Wing 820 825 830 Leu Gly Ser Phe 835

<210> 11

<211> 288

<212> DNA

<213> homo sapiens

<400> 11 gagtgcgggc acatcagtgt ctcagccccc atcgtccacc tgggggatcc catcacagcc 60

tcctgcatca tcaagcagaa ctgcagccat ctggacccgg agccacagat tctgtggaga 120

ctgggagcag agcttcagcc cgggggcagg cagcagcgtc tgtctgatgg gacccaggaa 180

tctatcatca ccctgcccca cctcaaccac actcaggcct ttctctcctg ctgcctgaac 240

tggggcaaca gcctgcagat cctggaccag gttgagctgc gcgcaggc 288

<210> 12

<211> 96

<212> PRT

<213> homo sapiens

<400> 12

Glu Cys Gly His Ile Ser Val Ser Ala Pro Ile Val His Leu Gly Asp 15 10 15

Pro Ile Thr Ala Ser Cys Ile Ile Lys Gln Asn Cys Ser His Leu Asp 20 25 30

Pro Glu Pro Gln Ile Leu Trp Arg Leu Gly Ala Glu Leu Gln Pro Gly 35 40 45

Gly Arg Gln Gln Arg Leu Ser Asp Gly Thr Gln Glu Ser Ile Ile Thr 50 55 60

Leu Pro His Leu Asn His Thr Gln Ala Phe Leu Ser Cys Cys Leu Asn 65 70 75 80

Trp Gly Asn Ser Leu Gin Ile Leu Asp Gln Val Glu Leu Arg Ala Gly 85 90 95

<210> 13

<211> 20

<212> DNA

<213> artificial

<220>

<223> sCSF3R-F1

<400> 13 atggcaaggc tgggaaactg 20

<210> 14

<211> 21

<212> PRT

<213> artificial

<220>

<223> sCSF3R-R1

<400> 14

Thr Thr Thr Wing Gly Thr Wing Gly Wing Gly Gly Cys Gly Gly Gly Gly 15 10 15

Thr Thr Thr Cys Gly 20

<210> 15

<211> 22

<212> DNA

<213> artificial

<220>

<223> nested sCSF3R-F1

<400> 15 acttgggctg ccctgatcat cc 22

<210> 16

<211> 22

<212> DNA

<213> artificial

<220>

<223> scFS3R-R1 nested

<400> 16 tggccaggct gatcatgaac tc 22

<210> 17

<211> 33

<212> DNA

<213> artificial

<220>

<223> scFS3R-EX1

<400> 17 gaaactgcag cctgacttgg gctgccctga tca 33

<210> 18 <211> 40

<212> DNA

<213> artificial

<220>

<223> sCSF3R-EX2

<400> 18 gaggcggggt ttcgccttgc tggccaggct gatcatgaac

<210> 19

<211> 31

<212> DNA

<213> artificial

<220>

<223> sCSF3R-EX3

<400> 19 atggcaaggc tgggaaactg cagcctgact t

<210> 20

<211> 38

<212> artificial <213> DNA

<220>

<223> sCSF3R-EX4

<400> 20 atttttttgt atttttagta gaggcggggt ttcgcctt

<210> 21

<211> 35

<212> DNA

<213> artificial

<220>

<223> sCSF3R-EX5

<400> 21 gcaggcttcg ccaccatggc aaggctggga aactg

<210> 22

<211> 37

<212> DNA

<213> artificial

<220>

<223> 5CSF3R-EX6

<400> 22 ggggacaagt ttgtacaaaa aagcaggctt cgccacc

<210> 23

<211> 37

<212> DNA

<213> artificial

<220>

<223> GCP sense

<400> 23 ggggacaagt ttgtacaaaa aagcaggctt cgccacc

<210> 24

<211> 51

<212> DNA

<213> artificial

<220>

<223> Reverse GCP

<400> 24 ggggaccact ttgtacaaga aagctgggtt tcaatggtga tggtgatggt g

<210> 25

<211> 20

<212> DNA

<213> artificial

<220>

<223> pEAK12F

<400> 25 gccagcttgg cacttgatgt

<210> 26

<211> 20

<212> DNA

<213> artificial

<220>

<223> pEAK12R

<400> 26 gatggaggtg gacgtgtcag

<210> 27

<211> 18

<212> DNA

<213> artificial

<220>

<223> 21M13

<400> 27 tgtaaaacga cggccagt

<210> 28

<211> 18

<212> DNA

<213> artificial

<220>

<223> M13REV

<400> 28 caggaaacag ctatgacc

<210> 29

<211> 19

<212> DNA

<213> artificial

<220>

<223> T7

<400> 29 taatacgact cactatagg

19

5

<210> 30 <211> 18 <212> Artificial DNA <213>

<220> <223> T3

10

<400> 30 attaaccctc actaaagg 18

Claims (17)

one.

A variant of the soluble isolated polypeptide of the CSF3R, wherein said polypeptide variant comprises or consists of a sequence of a ligand binding domain, lacks a transmembrane functional domain and lacks a cytoplasmic domain, where said ligand binding domain is a type domain immunoglobulin of the CSF3R, wherein said polypeptides further include following the immunoglobulin type domain amino acid residues 97-165 of SEQ ID NO: 4 or a fragment of at least 50 amino acids of amino acid residues 97-165 of SEQ ID NO : 4 and where said polypeptides retain the ability to bind the natural ligands of CSF3R and are further characterized in that the variant of the isolated soluble polypeptide of CSF3R is selected from SEQ ID NO: 2, a mature form of SEQ ID NO: 2 , a glycosylated form of SEQ: ID NO: 2, a pegylated form of SEQ ID NO: 2 or a functional equivalent of SEQ ID NO: 2, where the functional equivalent It has at least more than 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 2

2.

The variant of the soluble isolated polypeptide of the CSF3R of claim 1, wherein said mature form is SEQ ID NO: 4.

3.

The variant of the soluble isolated polypeptide of the CSF3R of claims 1 to 2, characterized in that the variant is a soluble variant of natural origin.

Four.

The variant of the soluble isolated polypeptide of the CSF3R according to claim 2, wherein said variant is selected from its glycosylated form, its pegylated form or its functional equivalent, where the functional equivalent has at least 75% identity, preferably at minus 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 4.

5.

A fusion protein comprising a soluble CSF3R polypeptide of any one of claims 1 to 4 operatively linked to an additional amino acid domain.

6.

The fusion protein of claim 5, wherein said additional amino acid domain comprises a signal peptide, a marker, a targeted peptide, the constant domain of an immunoglobulin, a multidimerization domain or its biologically active protein or a fragment thereof.

7.

The fusion protein of claim 6, comprising a CSF3R polypeptide of any one of claims 1 to 4 operatively linked to the constant domain of an immunoglobulin.

8.

A conjugate comprising a CSF3R polypeptide of any one of claims 1 to 4 or a fusion protein of any one of claims 5 to 7.

9.

An isolated nucleic acid molecule encoding a polypeptide of any one of claims 1 to 7.

10.

The isolated nucleic acid molecule of claim 9 which is a cDNA molecule.

eleven.

The isolated nucleic acid molecule of claim 9 or claim 10 comprising or consisting of a nucleotide sequence selected from SEQ ID NO: 1, SEQ 10 NO: 3, SEQ 10 NO: 5, SEQ 10 NO: 7, or a complementary strand or its degenerate sequence.

12.

A vector comprising a nucleic acid molecule of any one of claims 9 to 11.

13.

A recombinant host cell, wherein said cell comprises a nucleic acid molecule of any one of claims 9 to 11 or a vector of claim 12.

14.

The host cell of claim 13, which expresses a polypeptide of any one of claims 1 to 7.

fifteen.

The host cell of claim 13 or claim 14, which is a prokaryotic or eukaryotic cell.

16.

A method for producing a polypeptide of any one of claims 1 to 7, a method comprising culturing a recombinant host cell of any one of claims 13 to 15 under conditions that allow the expression of the nucleic acid molecule and recovering the polypeptide produced.

17.

A pharmaceutical composition comprising a polypeptide variant of any one of claims 1 to 4, a fusion protein of any one of claims 5 to 7, a conjugate of claim 8 and a pharmaceutically acceptable carrier or diluent.